Methods and compositions for treating mucosal tissue disorders

ABSTRACT

The present invention provides compositions and formulations comprising glutathione with or without thiocyanate and methods of use thereof to treat diseases and disorders in mucosal/epithelial tissue.

STATEMENT OF PRIORITY

This application is a continuation application under 35 U.S.C. §111(a)of, and claims priority to, PCT Application No. PCT/US2013/067307, filedOct. 29, 2013, which claims the benefit, under 35 U.S.C. §119(e), ofU.S. Provisional Application Ser. No. 61/719,804, filed Oct. 29, 2012,the entire contents of each of which are incorporated by referenceherein.

FIELD OF THE INVENTION

The present invention generally relates to pharmaceutical compositionscomprising glutathione, ascorbate and bicarbonate with or withoutthiocyanate and methods of use thereof to treat diseases and disordersin mucosal tissue.

BACKGROUND OF THE INVENTION

Mucosal surfaces (such as the oral cavity, eye, gastrointestinal,urogenital, and respiratory tracts) by design interface with potentiallynoxious environments. Protection against pathologic consequences of thisdirect environmental encounter is highly dependent on the exocrinesecretions that bathe these exposed surfaces. These secretions providethe first line of defense against a variety of insults by providingphysical barriers, mechanical clearance, and targeted pathogenneutralization without bystander damage of host tissues and consequentloss of function. To this end, these secretions deliver a variety ofsoluble factors including macromolecules, low molecular weight moleculesand ions that work synergistically to maintain a healthy homeostasis bylimiting host tissue exposure to pathogens and minimizing the hostdestructive consequences of inappropriate inflammatory responses andassociated oxidative and nitrosative stresses.

For example, chronic lung and airway diseases are often characterized bya loss of homeostatic balance resulting in refractory infections and anorgan-destructive host inflammatory response to exogenous stimuli.Pharmacologic doses of hormones such as glucocorticoids, otherimmunosuppressant therapies and antibiotics can provide some clinicalrelief, but are often inadequate. Alternatively, genetic informationidentifying specific protein dysfunction in disease suggests othertherapeutic approaches for heritable diseases such as gene therapy forcystic fibrosis (CF). However, a need still exists for more adequate andeffective treatment of such disorders.

The present invention overcomes previous shortcomings in the art byproviding pharmaceutical compositions comprising glutathione, ascorbateand bicarbonate with or without thiocyanate and methods of use thereofto treat diseases and disorders in mucosal/epithelial tissue.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a composition comprisinga) glutathione, a pharmaceutically-acceptable salt of glutathione, or aderivative or a prodrug thereof, b) an organic acid, apharmaceutically-acceptable salt thereof (e.g., ascorbic acid), or aderivative or prodrug thereof, and c) a bicarbonate salt, such as sodiumor potassium bicarbonate.

It is to be understood that in referring to a composition of thisinvention, wherein the terms glutathione, organic acid, or ascorbic acidare used without also referring to a pharmaceutically acceptable salt,derivative or prodrug thereof, such pharmaceutically acceptable salt,derivative or prodrug thereof is intended.

A pH adjusting agent can also be present in the compositions of thisinvention, if necessary or desired, to adjust the pH of the compositionin some embodiments to be within a range from about 5 to about 9, insome embodiments to be within a range from about 6 to about 8, and insome embodiments to be within a range from about 6.5 to about 7.5. Inone embodiment, the pH adjusting agent can be a bicarbonate salt.

It is to be understood that, as the relative amounts of glutathione andorganic acid, such as ascorbic acid, can vary, the amount of bicarbonatesalt can also vary. If glutathione and the organic acid are mixed with abicarbonate salt, the acid functional groups will react with thebicarbonate to form the salts of the glutathione and the organic acid,and the bicarbonate will be acidified to form carbonic acid, withconcomitant formation of water and evolution of carbon dioxide.Accordingly, if the formulation is prepared by mixing the acids with abicarbonate salt, what is intended is that sufficient bicarbonate isadded wherein the bicarbonate salt is present in a molar amount equal tothe combined molar amount of the glutathione and the organic acid or ina molar excess of the combined molar amount of the glutathione and theorganic acid. For example, the molar excess of the bicarbonate salt canbe greater than 1.0 and less than about 1.5.

The weight ratios of the components of the compositions of thisinvention can vary over a wide range. Typically, the amount of eachcomponent is selected such that there is sufficient bicarbonate salt inthe composition to bring the pH of the composition to the desired range,while also allowing for the bicarbonate salt to be present along withthe glutathione and organic acid.

For example, the amount of (a) the glutathione, apharmaceutically-acceptable salt of glutathione, or a derivative or aprodrug thereof can be from about 0.1 to about 95 percent by weight, theamount of (b) the organic acid, a pharmaceutically-acceptable saltthereof or a derivative or prodrug thereof can be from about 0.5 toabout 60 percent by weight, and the weight of (c) the bicarbonate saltcan be from about 1 to about 55 percent by weight. Within these ranges,the amount of (a) can be, for example, from about 52 to about 80 weightpercent, (b) can be from about 3 to about 18 weight percent, and (c) canbe from about 17 to about 30 weight percent.

In some embodiments, the compositions can further comprise from about0.01% to about 5% by weight of a pharmaceutically-acceptable thiocyanatesalt, such as from about 1% and about 2% by weight of a thiocyanatesalt, while maintaining the weight ratios of the other components.

In some embodiments, the compositions can further include precursors toglutathione, such as methionine, cysteine and/or N-acetyl cysteine, inamounts from about 0.1% to about 10% by weight, while maintaining theweight ratios of the other components. In one aspect of this embodiment,glutathione precursors such as, methionine, cysteine and/or N-acetylcysteine can be used in place of glutathione, rather than in addition toglutathione. In this aspect, the amount of the glutathione precursor inthe composition can be that of glutathione.

In some embodiments, the present invention provides a pharmaceuticalcomposition comprising about 0.1 to about 95 weight percent glutathione,a pharmaceutically acceptable salt thereof, a derivative thereof, ananalogue thereof and/or a prodrug thereof, about 0.5 to about 60 weightpercent organic acid, a pharmaceutically acceptable salt thereof, aderivative thereof, an analogue thereof and/or a prodrug thereof, andabout 1 to about 55 weight percent bicarbonate. In some embodiments,this pharmaceutical composition can further comprise about 0.01 to about20 weight percent thiocyanate, a pharmaceutically acceptable saltthereof, a derivative thereof, an analogue thereof and/or a prodrugthereof.

Also provided herein is a pharmaceutical composition comprising about 1to about 14 weight percent glutathione, a pharmaceutically acceptablesalt thereof, a derivative thereof, an analogue thereof and/or a prodrugthereof, about 0.1 to about 5 weight percent organic acid, apharmaceutically acceptable salt thereof, a derivative thereof, ananalogue thereof and/or a prodrug thereof, about 0.1 to about 10 weightpercent bicarbonate and about 70 to about 85 weight percent water.

Further provided herein is a pharmaceutical composition comprising about1 to about 14 weight percent glutathione, a pharmaceutically acceptablesalt thereof, a derivative thereof, an analogue thereof and/or a prodrugthereof, about 0.1 to about 5 weight percent organic acid, apharmaceutically acceptable salt thereof, a derivative thereof, ananalogue thereof and/or a prodrug thereof, about 0.1 to about 10 weightpercent bicarbonate, about 0.01 to about 2 weight percent thiocyanate, apharmaceutically acceptable salt thereof, a derivative thereof, ananalogue thereof and/or a prodrug thereof and about 15 to about 85weight percent water.

The present invention additionally provides a pharmaceutical compositioncomprising glutathione, a pharmaceutically acceptable salt thereof, aderivative thereof, an analogue thereof and/or a prodrug thereof,ascorbic acid, a pharmaceutically acceptable salt thereof, a derivativethereof, an analogue thereof and/or a prodrug thereof, and sodiumbicarbonate in a weight to weight to weight ratio of about 1.5:1:0.8 to95:1:4.

Further provided herein is a pharmaceutical composition comprisingglutathione, a pharmaceutically acceptable salt thereof, a derivativethereof, an analogue thereof and/or a prodrug thereof, ascorbic acid, apharmaceutically acceptable salt thereof, a derivative thereof, ananalogue thereof and/or a prodrug thereof, sodium bicarbonate, andthiocyanate, a pharmaceutically acceptable salt thereof, a derivativethereof, an analogue thereof and/or a prodrug thereof in a weight toweight to weight to weight ratio of about 1:90:100:1 to 1000:10:40:1.

Additionally provided herein is a pharmaceutical composition comprisingglutathione, a pharmaceutically acceptable salt thereof, a derivativethereof, an analogue thereof and/or a prodrug thereof, an organic acid,a pharmaceutically acceptable salt thereof, a derivative thereof, ananalogue thereof and/or a prodrug thereof, a bicarbonate and water in aweight to weight to weight to weight ratio of about 1:60:60:600 to about100:1:1:20.

The present invention also provides a pharmaceutical compositioncomprising glutathione, a pharmaceutically acceptable salt thereof, aderivative thereof, an analogue thereof and/or a prodrug thereof, anorganic acid, a pharmaceutically acceptable salt thereof, a derivativethereof, an analogue thereof and/or a prodrug thereof, a bicarbonate,thiocyanate, a pharmaceutically acceptable salt thereof, a derivativethereof, an analogue thereof and/or a prodrug thereof and water in aweight to weight to weight to weight to weight ratio of about1:100:100:1:1,000 to about 1,000:10:40:1:250.

In further embodiments of the pharmaceutical compositions recited above,the compositions can include a pH adjusting agent, which can be, but isnot limited to, citric acid, sodium citrate, sodium bicarbonate, dibasicsodium phosphate, magnesium oxide, calcium carbonate and magnesiumhydroxide, an acetate buffer, a citrate buffer, a phosphate buffer, alactic acid buffer, a borate buffer and any combination thereof.

In further embodiments, the present invention provides a pharmaceuticalcomposition comprising glutathione, ascorbic acid and sodium bicarbonatein a weight to weight to weight ratio of about 4.13:1:1.74 to 20:1:4.57.

Also provided herein is a pharmaceutical composition comprisingglutathione, ascorbic acid, sodium bicarbonate and water in a weight toweight to weight to weight ratio of about 4.13:1:1.73:16.39 to19.96:1:4.55:88.18.

The present invention additionally provides a pharmaceutical compositioncomprising about 52 to about 78 weight percent glutathione, about 3.5 toabout 18 weight percent ascorbic acid, about 17 to about 30 weightpercent sodium bicarbonate, and about 0.2 to about 1.0 weight percentthiocyanate.

Additional embodiments of this invention include a pharmaceuticalcomposition comprising about 52 to about 80 weight percent glutathione,about 3 to about 18 weight percent ascorbic acid or about 17 to about 30weight percent sodium bicarbonate.

Furthermore, the present invention provides a pharmaceutical compositioncomprising glutathione, ascorbic acid, sodium bicarbonate andthiocyanate in a weight to weight to weight to weight ratio of about220:11.11:50.78:1 to 220:53.78:93.33:1.

Also provided herein is a pharmaceutical composition comprising about 17to about 19 weight percent glutathione, about 0.5 to about 4.5 weightpercent ascorbic acid, about 4 to about 8 weight percent sodiumbicarbonate, about 0.05 to about 0.5 weight percent thiocyanate andabout 70 to about 81 weight percent water.

Also provided herein is a pharmaceutical composition comprising about 17to about 20 weight percent glutathione, about 0.5 to about 5 weightpercent ascorbic acid, about 4 to about 8 weight percent sodiumbicarbonate and about 65 to about 81 weight percent water.

In addition, the present provides a pharmaceutical compositioncomprising glutathione, ascorbic acid, sodium bicarbonate, thiocyanateand water in a weight to weight to weight to weight to weight ratio ofabout 220:11:50:1:975 to 220:53:93:1:900.

A further aspect of the invention is a pharmaceutical compositioncomprising 72.92% by weight glutathione, 4.86% by weight ascorbic acid,and 22.22% sodium bicarbonate.

Also provided herein is a pharmaceutical composition comprising 53.12%by weight glutathione, 17.14% by weight ascorbic acid, and 29.74% byweight sodium bicarbonate.

The present invention further provides a pharmaceutical compositioncomprising 13.96% by weight glutathione, 0.93% by weight ascorbic acid,4.25% by weight sodium bicarbonate, and 80.86% water.

A pharmaceutical composition is also provided herein, comprising 13.51%by weight glutathione, 4.36% by weight ascorbic acid, 7.57% by weightsodium bicarbonate, and 74.56% by weight water.

The present invention additionally provides a pharmaceutical compositioncomprising 78.22% by weight glutathione, 3.91% by weight ascorbic acid,and 17.87% by weight sodium bicarbonate.

Further provided herein is a pharmaceutical composition comprising60.17% by weight glutathione, 14.56% by weight ascorbic acid, and 25.27%sodium bicarbonate.

An additional embodiment of this invention provides a pharmaceuticalcomposition comprising 18.36% by weight glutathione, 0.92% by weightascorbic acid, 4.19% by weight sodium bicarbonate, and 76.53% water.

Furthermore, the present invention provides a pharmaceutical compositioncomprising 17.77% by weight glutathione, 4.3% by weight ascorbic acid,7.46% by weight sodium bicarbonate, and 70.47% by weight water.

Additionally provided herein is a pharmaceutical composition comprising0.78% by weight glutathione, 46.56% by weight ascorbic acid, 52.65% byweight sodium bicarbonate.

In yet further embodiments, the present invention provides apharmaceutical composition comprising 0.13% by weight glutathione, 8.12%by weight ascorbic acid, 9.18% by weight sodium bicarbonate, and 82.57%by weight water.

A further aspect of the invention is a pharmaceutical compositioncomprising 72.92% by weight glutathione, 4.86% by weight ascorbic acid,and 22.22% sodium bicarbonate.

Also provided herein is a pharmaceutical composition comprising 53.12%by weight glutathione, 17.14% by weight ascorbic acid, and 29.74% sodiumbicarbonate.

The present invention further provides a pharmaceutical compositioncomprising 13.96% by weight glutathione, 0.93% by weight ascorbic acid,4.25% by weight sodium bicarbonate, and 80.86% water.

A pharmaceutical composition is also provided herein, comprising 13.51%by weight glutathione, 4.36% by weight ascorbic acid, 7.57% by weightsodium bicarbonate, and 74.56% by weight water.

The present invention additionally provides a pharmaceutical compositioncomprising 78.22% by weight glutathione, 3.91% by weight ascorbic acid,and 17.87% by weight sodium bicarbonate.

Further provided herein is a pharmaceutical composition comprising60.17% by weight glutathione, 14.56% by weight ascorbic acid, and 25.27%sodium bicarbonate.

An additional embodiment of this invention provides a pharmaceuticalcomposition comprising 18.36% by weight glutathione, 0.92% by weightascorbic acid, 4.19% by weight sodium bicarbonate, and 76.53% water.

Furthermore, the present invention provides a pharmaceutical compositioncomprising 17.77% by weight glutathione, 4.3% by weight ascorbic acid,7.46% by weight sodium bicarbonate, and 70.47% by weight water.

Additionally provided herein is a pharmaceutical composition comprising0.78% by weight glutathione, 46.56% by weight ascorbic acid, 52.65% byweight sodium bicarbonate.

In yet further embodiments, the present invention provides apharmaceutical composition comprising 0.13% by weight glutathione, 8.12%by weight ascorbic acid, 9.18% by weight sodium bicarbonate, and 82.57%by weight water.

A composition of this invention can be present, for example, as a solidformulation, such as a particle formulation, or as a solution. When in aparticle formulation, the particles can be mixed with gases, or liquidpropellants, for use in inhalation therapy. Other solid formulationsinclude formulations for oral administration, buccal administration orcolonic administration, and suppositories for rectal or vaginaladministration. Exemplary formulations include, but are not limited to,the following: eye drops, nebulizers, topical gels and ointments, drypowders, particles, sprays, liquids, anesthetic machines or vaporizers,autoinjectors, intrauterine devices, respimats, liniments, liposomes,lotions, formulations for intramuscular, intrathecal, or subcutaneousinjection, douches, infusions, and face masks.

In solution form, the formulations can be in the form of sprays forintranasal administration, formulations for use in nebulizers, andformulations for rectal administration, such as enemas and colonies.Solutions that include water-miscible organic solvents, such aspropylene glycol and/or glycerol, and other components normally found invaginal and rectal lubricants, can also be used. Regardless of thesolvents used, the solvent is typically present in a weight ratio offrom about 15 to about 85 percent by weight, relative to the weight ofthe solids, and, more typically, is from about 50 to about 85% byweight.

The compositions and/or formulations of this invention can be used totreat disorders associated with a mucosal membrane, by delivering thecompositions and/or formulations to the mucosal membrane(s) to betreated. In some embodiments, the mucosal membrane can be in or near thelungs, such as the deep lung (alveolar region), and in otherembodiments, the mucosal membrane(s) can be in or near one or more ofthe eyes, mouth, nose, rectum, and/or vagina.

In still further embodiments, the composition of this invention canfurther comprise, and/or the composition can be administered incombination or in alternation with, one or more therapeutic agents. Insome embodiments, the composition of this invention and therapeuticagent(s) are directed to the same locus in the same formulation, and inother embodiments, the composition can be administered via one pathway,and the therapeutic agent(s) can be administered via a differentpathway.

In some embodiments, the therapeutic agent(s) treat the disorder forwhich they are administered, but cause certain side effects, such as adrying of a mucosal membrane that results in discomfort and/or injury,which side effects can be addressed by administering a composition ofthis invention to a subject in need thereof.

In other embodiments, the therapeutic agent and composition of thisinvention both treat the underlying disorder, though via differentmeans, such that an additive or synergistic effect can be achieved. As aresult, in some aspects of this embodiment, a lower dose of thetherapeutic agent can be effective, which lower dose can result in fewerside effects, or provide other benefits to the subject. By lower dose ismeant a dose that is less than a dose that would typically beadministered in the absence of administration of a composition of thisinvention.

In embodiments of this invention that comprise a therapeutic agent, theresulting new formulation can provide a new application for thetherapeutic agent, enhance the efficacy of the therapeutic agent, reduceunwanted side effects associated with the therapeutic agent, and/orreduce the dose of the therapeutic agent.

Exemplary therapeutic agents of this invention include, but are notlimited to, monoclonal antibodies; immunomodulatory agents, includingagents that cause T and B cell activation, proliferation, and/ormaturation; agents that bring about innate immune system activation,proliferation and/or maturation (e.g., INK, MAPK, ERK, NK kappa Bpathway agonists or antagonists, and monocyte, neutrophil, or macrophageagonists or antagonists); matrix metalloproteinase inhibitors; heatshock protein agonists or antagonists; alpha synuclein inhibitors;chelating agents; diuretics; alpha 1 antitrypsin modulators;purinoceptor agonists or antagonists; cyclooxygenase 2 inhibitors; DNAgyrase inhibitors; natural killer cell and natural killer T cellagonists or antagonists; cathepsin class agonists or antagonists;antioxidant therapy agents; rho-associated kinase inhibitors; myosininhibitors; phosphatidylinositol 3 kinase inhibitors and relatedmolecules; nitric oxide synthase agonists or antagonists; nitric oxideagonists or antagonists; ion channel function or trafficking modulators;surfactants, in particular, lung surfactants; cannabinoid receptormodulators; complement system inhibitors; IgE receptor antagonists; Gprotein-coupled receptor agonists or antagonists; chemokines; chemokinereceptor agonists or antagonists; cytokines; cytokine receptor agonistsor antagonists; arachidonic acid agonists or antagonists; inflammationmediators; STAT6 inhibitors; histamine or leukotriene agonists orantagonists; calcineurin agonists or antagonists; and ant combinationthereof.

In some embodiments wherein the composition of this invention includesone or more therapeutic agent, the composition can be used to treat amucosal disease or disorder a pulmonary disease or disorder, anautoimmune disease or disorder (e.g., multiple sclerosis, Crohn'sdisease, ulcerative colitis, lupus, inflammatory bowel syndrome,irritable bowel syndrome, etc.), an infectious disease (e.g., HIVinfection), a neurodegenerative disease or disorder (e.g., Alzheimer'sdisease), and the like, singly or in any combination.

In some embodiments of this invention, the one or more therapeuticagents can be present with the glutathione, organic acid and bicarbonatein a single formulation, and in other embodiments, the one or moretherapeutic agents can be present in a first formulation and theglutathione, organic acid and bicarbonate can be present in a secondformulation that is separate from the first formulation. As such, thecompositions and formulations can be used in combination or ‘kit’therapies. As one non-limiting example, the formulation with thetherapeutic agent can be present in oral, injectable, and/or inhaledforms, and the glutathione, organic acid and bicarbonate can be presentin an inhaled (e.g., pulmonary or intranasal) formulation.

While not wishing to be bound by any particular theory, in someembodiments of this invention, the formulations disclosed herein can beeffective in achieving and/or maintaining a normal lung mucosa, or atleast a more normal lung mucosa, which is an important factor inmaintaining lung health. Drugs administered to the lungs are oftenassociated with certain side effects, in some cases because of dosage,and in other cases because they damage the lung tissue. In someembodiments, therapeutic agents combined with the formulations disclosedherein are effective at lower doses, and at such lower doses, theincidence of side effects can be reduced. For example, one can decreaseinhaled corticosteroid (ICS) dosing and accordingly, reduce the risk ofpneumonia in a subject with chronic obstructive pulmonary disease(COPD), and one can reduce the dose of β-agonists and otherbronchodilators to reduce the risk of death in asthma patients.

In some embodiments, wherein the therapeutic agent interacts unfavorablywith lung tissue, the formulation of this invention can be administeredto restore homeostasis to the lung tissue, and thus minimize oreliminate side effects and/or damage caused by the therapeutic agent.

The present invention also provides a method of treating an airwaydisorder or disease in a subject in need thereof, comprising deliveringto the subject an effective amount of a composition of this invention,optionally in combination with a steroid and/or bronchodilator, such asa beta₂-agonist.

Furthermore, the present invention provides a method of treating aninfection in the airway of a subject in need thereof, comprisingdelivering to the subject an effective amount of a composition of thisinvention, optionally in combination with one or more antibiotics. Theantibiotics can be administered locally to the lungs and/orsystemically.

Inflammation in the airway of a subject in need thereof can be treatedby delivering to the subject an effective amount of a composition ofthis invention.

In addition to providing methods of treatment, the compositionsdescribed herein can be used to provide prevention and/or ameloriationof various diseases and disorders. For example, there are a number ofsmoking related disorders, such as COPD, cardiac disorders, urinarytract disorders including cancer, gastrointestinal disorders such aspeptic ulcer disease, and the like, which can be prevented, at least tosome extent, by administering a composition of this inventionprophylactically. Prophylactic administration of a composition of thisinvention to mucosal tissue can establish and/or maintain homeostasis inthe mucosal tissue, to prevent and/or ameliorate damage or otherpathological harm to the tissue.

A composition of this invention can also be administeredprophylactically during cold and flu season, to prevent, ameliorate orreduce the severity of a viral infection in the lungs and other mucosalmembranes. This is particularly true of medical workers, who are exposedto a variety of infectious agents, including viruses, bacteria, fungi,and the like. Maintenance of homeostasis in mucosal tissue helps toprotect against infectious diseases, so prophylactic administration isparticularly useful for such workers.

A subject in a polluted or contaminated air environment can be protectedagainst the effects of air pollution or contamination on the lungs andother mucosal membranes by prophylactically administering a compositionof this invention to the mucosal membrane(s) to be protected. Forexample, medical professionals, soldiers, fire fighters, factoryworkers, and the like that work in an air-polluted and/or contaminatedenvironment (e.g., radon contamination) can benefit from prophylacticadministration of the compositions of this invention.

Prophylactic administration of a composition of this invention can alsobe useful when a subject is exposed to dry air and/or otherairway-irritating environments, such as those experienced when flying.

Yet further embodiments of this invention include the use of acomposition of this invention in the manufacture of a medicament fortreating a mucosal tissue disease or disorder, treating an infectionan/or a disease or disorder caused by infection by a pathogen in mucosaltissue and/or treating inflammation in mucosal tissue in a subject.

Also provided herein is the use of a composition of this invention inthe manufacture or preparation of a medicament for treating a disorderof the mouth, treating a pulmonary or airway disorder or disease,treating a pulmonary or airway disorder or disease, wherein thepulmonary or airway disorder is an inflammatory disorder, treating adisorder of the eye, treating a disorder of the central nervous system,treating a nasal disorder, treating a gastrointestinal disorder,treating a urinary tract disorder, renal disorder, and/or disorder inthe urogenital tract, treating a disorder of the circulatory system,restoring homeostasis to and/or maintaining homeostasis in a mucosalmembrane of a subject with cystic fibrosis or a lung transplant,reducing dryness in a mucosal membrane of a subject treated with ananticancer agent that causes dryness in the mucosal membrane, preservingan organ for organ transplantation, preventing a lung disease ordisorder caused by infection by a pathogen, preventing and/or reducingthe symptoms of a gastrointestinal disorder with an inflammatorycomponent, and restoring homeostasis to or maintaining homeostasis in avaginal and/or rectal mucosal membrane, singly or in any combination.

The foregoing and other aspects of the present invention will now bedescribed in more detail with respect to other embodiments describedherein. It should be appreciated that the invention can be embodied indifferent forms and should not be construed as limited to theembodiments set forth herein. Rather, these embodiments are provided sothat this disclosure will be thorough and complete, and will fullyconvey the scope of the invention to those skilled in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-B. Glutathione inhibits nitrate dependent growth. Pseudomonasaeruginosa strain PAO1 is capable of growing in the absence of oxygenusing nitrate for respiration. The presence of nitrate to 1% in LauriaBertani (LB) broth had no discernible effect on growth in ambientatmosphere and this strain was incapable of growth in LB broth in ananaerobic atmosphere of 5% CO₂-10% H₂-85% N₂ without supplementation.The studies presented in FIG. 1 examine whether glutathione influencesthe growth of PAO1 either in the presence or absence of nitrate undereither ambient (aerobic) or anaerobic atmospheres. There were nodiscernible differences between the influences of glutathione on theaerobic growth of PAO1 either in the presence or absence of 1% nitratein LB broth (A). There was a trend toward reduced growth yield at 14 hwith increasing concentrations of glutathione that reached significancewith 7.5 mM glutathione consistent with the reducing potential of theglutathione limiting oxygen dependent growth. In contrast, there wastotal inhibition of nitrate dependent growth (B) with the 5 and 7.5 mMconcentrations of glutathione and no discernible effects with 2.5 mM orless.

FIG. 2. Glutathione potentiation of nitrite mediated inhibition of PAO1while blocking H₂O₂ mediated inhibition. Previous studies indicated thatwith nitrate (NO₃) dependent respiration that the resulting products ofnitrite (NO₂) and nitric oxide (NO) were responsible for inhibition ofbacterial growth. It was further suggested that oxygen dependent growthcan proceed in the presence of nitrate, but was inhibited by millimolarconcentrations of either nitrite or nitric oxide. The fact thatglutathione was inhibitory for oxygen depleted, nitrate-dependentgrowth, but not for aerobic growth in the presence of nitrate (FIG. 1),suggested the inhibitory effects of glutathione were dependent onnitrate reduction, suggesting that the inhibitory effects of nitrite andnitric oxide previously observed might be potentiated by glutathione. Totest this possibility, reduced glutathione was titrated vs. increasingconcentrations of KNO₂ in either trypticase soy broth (TSB) in ambientatmosphere or in trypticase soy broth with nitrate (TSBN) (1% KNO₃)under anaerobic atmosphere. Reduced glutathione resulted in a slight,but significant, concentration-dependent increase in growth yield in theabsence of nitrite. In these experiments, nitrite to 14 mM had nodiscernible inhibition on aerobic growth in the absence of glutathione(FIG. 2). There was synergistic, concentration-dependent inhibitionbetween glutathione and nitrite, resulting in significant inhibitionwith 6.4 mM GSH and 14 mM KNO₂ and with 12.8 mM GSH and 7 mM KNO₂. Incontrast, increasing the concentration of GSH protected PAO1 from thegrowth inhibiting effects of glucose oxidase, consistent with reducedglutathione dependent bacterial peroxidase activity. Nitrate wasrequired for PAO1 growth in TSB under anaerobic atmosphere and theaddition of KNO₂ at the concentrations tested in these experimentsresulted in significant inhibition even in the absence of glutathione(not shown). There was, however, further inhibition evident withincreasing concentrations of GSH in the presence of nitriteconcentrations that did not result in total inhibition by themselves.

FIGS. 3A-B. Glutathione potentiation of nitrite inhibition isindependent of the redox state of the glutathione. These studiesexamined the question of the importance of the redox state ofglutathione to its contributions to the inhibition by RNI and to theblocking of the glucose oxidase antimicrobial effects. Glutathione ineither its reduced (GSH) or oxidized (GSSG) form was titrated in thepresence or absence of 14 mM nitrite in TSB and PAO1 was grown inambient atmosphere. Both GSH and GSSG at molar equivalency resulted ininhibition of PAO1 growth in the presence of nitrite (A). In contrast,GSH but not GSSG protected PAO1 from inhibition with the glucose oxidasesystem (B). Additional studies indicated that growth inhibition of PAO1by exogenous H₂O₂ was also blocked by 14 mM GSH, suggesting a directeffect on H₂O₂ activity and not the indirect blocking of the enzymaticactivity of glucose oxidase. These data suggest that the oxidized formof glutathione would be efficacious in inhibiting nitrate dependentgrowth without the possible adverse consequence of protecting thebacteria from host innate defenses dependent upon H₂O₂ antibacterialactivity.

FIG. 4. Glutathione enhances S-nitrosoglutathione inhibition of nitratedependent growth of PAO1. Previous studies suggested that the nitricoxide donor S-nitrosoglutathione (GSNO) was capable of inhibitingnitrate-dependent growth of P. aeruginosa. This raised the possibilitythat the potentiating effects of glutathione on the antimicrobialactivity of nitrate metabolites were the result of generation of themore active nitrosoglutathione specie. This would suggest thepossibility that glutathione would have little additional effect on theactivity of authentic nitrosoglutathione. To examine this possibility,S-nitrosoglutathione (Sigma) was tested for influence on growth of PAO1in TSBN (1% KNO₃) with and without 6.4 mM glutathione. There was aconcentration dependent inhibition of PAO1 growth as determined bybacterial density (A_(□650nm)) at 18 h. This inhibition wassignificantly enhanced in the presence of glutathione with anexponential decrease in the concentration of GSNO necessary for totalinhibition of growth achieved at 18 h.

FIG. 5. Glutathione enhances the inhibitory activity of NO gas of PAO1aerobic growth. Previous studies demonstrated that NO gas was capable ofinhibiting PAO1 aerobic growth and suggested that NO was the ultimateproduct of nitrate metabolism responsible for the shutdown in bacterialgrowth. This would suggest the potentiating effects of glutathioneshould be evident with NO gas. Discrete volumes of TSB were surfacegassed with 2.31% NO in N₂ gas mixture for a fixed time (45 min) thatresulted in a two-fold titration from a concentration that resulted inminimal inhibition of aerobic growth. There was dose dependentenhancement of inhibition at the 0.5 relative concentration of NO byGSH, but not at lower NO concentrations. The same experiment designed toexamine the influence of NO on anaerobic growth in TSBN did not work,presumably because the process of degassing and reducing O₂ for entryinto the anaerobic chamber reduced the NO in the media. This was trueeven if GSH was available in the media before degassing, suggesting thatthe NO was not stabilized by interaction with GSH.

FIG. 6. Glutathione potentiation of NO mediated inhibition of Pa PAO1aerobic growth in Luria Bertani broth is independent of order ofaddition of GSH. If NO reacts with glutathione to generatenitrosoglutathione then gassing with NO in the presence of glutathionemay be expected to yield greater activity than gassing first and thenadding glutathione. To test this effect, LB broth was either pre-gassedwith NO followed by titration of GSH or the LB broth aliquots containingvarious concentrations of GSH were gassed. Titration of NO wasaccomplished by varying the gassing times of the LB broth. There was nodiscernible inhibition with any of the concentrations of NO in theabsence of glutathione. In the presence of 5 mM glutathione, the lowestconcentration of NO (10 minutes gassing time) resulted in totalinhibition. The 2.5 mM revealed a dose dependent NO inhibition ofgrowth. The growth inhibiting synergistic activity between glutathioneand NO was independent of whether glutathione was present during gassing(NO-GSH) or added after (NO-LB+GSH).

FIGS. 7A-B. Complementary effects of ascorbate on glutathione inhibitionof nitrate independent and dependent growth of PAO1. The next series ofexperiments was designed to determine if there were any influences ofascorbate on glutathione potentiation of the growth inhibition of theRNI (FIGS. 7, 8 and 9). Again aerobic growth was minimally influenced bythe presence of nitrate in the absence of both glutathione and ascorbateand there were no discernible differences on the effects of ascorbatetitration on growth in the presence or absence of nitrate (A). Incontrast, there was a dose dependent reduction in the aerobic growthattained by PAO1 that could be attributed to glutathione in LB broththat was further enhanced by adding increasing concentrations ofascorbic acid (A). The most pronounced inhibition in the absence ofnitrate of aerobic growth occurred with 7.5 mM glutathione that wasenhanced by titration of ascorbate reaching total inhibition with 2.1 mMascorbate (A). This was likely due to the combined reducing potential ofthe glutathione and ascorbate limiting oxygen dependent growth. Incontrast, ascorbate concentrations of 0.2 mM with 7.5 mM glutathioneresulted in profound inhibition of growth in ambient atmosphere in thepresence of nitrate. Likewise, the presence of nitrate resulted ingreater inhibition of aerobic growth with 5 mM glutathione andascorbate. In the absence of glutathione, the influence of ascorbate ongrowth in ambient atmosphere appeared to be independent of the presenceof nitrate (A). In contrast, growth in an anaerobic atmosphere was onlyevident with nitrate. Only the 2.5 mM (B) and lower concentrations ofglutathione permitted growth in the absence of ascorbate. There was adose dependent decrease in nitrate-dependent growth with increasingconcentrations of ascorbate in the absence of glutathione that was morepronounced in the presence of 2.5 mM glutathione (B).

FIG. 8. Ascorbate effects on the inhibitory activities of nitrite andglutathione on aerobic growth of PAO1. Ascorbate was titrated over acheckerboard titration of glutathione and KNO₂ in LB broth. The datapresented are representative of three different experiments with a widerrange of concentrations of individual components. In these experiments,neither KNO₂ to 3.5 mM, glutathione to 5 mM nor ascorbate to 2.1 mM haddiscernible effects on the aerobic growth of PAO1 attained by 17 h.There also was no discernible effect of ascorbate addition to eitherglutathione or KNO₂ at the concentrations tested. Again there wereconcentration dependent inhibitory effects for KNO₂ in the presence ofglutathione that were enhanced by the addition of ascorbate. Thissynergistic inhibition was evident with as little as 0.875 mM KNO₂ inthe presence of 5 mM glutathione and as little as 2.5 mM glutathione inthe presence of 1.75 mM KNO₂.

FIGS. 9A-D. Ascorbate effects on the inhibitory activities of nitricoxide and glutathione on aerobic growth of PAO1. In these experiments,varying concentrations of NO were achieved again by varying the gassingtime (10, 15, 30 or 45 min) with 2.3% NO in N₂ either in the presence(NO-GSH) or absence (NO-LB) of varying concentrations of glutathione.Glutathione was added to NO-LB in varying concentrations (NO-LB+GSH).Again in the absence of NO there were no inhibitory effects of eitherthe glutathione or the ascorbate concentrations and combinationsemployed in this study. Bubbling of the media with N₂ gas for 45 min didnot have any of the inhibitory effects of the NO—N₂ gas mixture. At thehighest concentration (5 mM) of glutathione presented there was totalinhibition of growth in the presence of the lowest concentration of NOregardless of the order of NO/glutathione addition (B). With the 2.5 mMconcentration of glutathione there may have been some advantage tobubbling in the presence of glutathione (2.5 mM NO-GSH) as opposed toadding glutathione after bubbling (NO-LB+2.5 mM GSH); however, anydifferences due to order of addition appeared to be lost with longer NOdelivery (B, C and D). With the 15 min delivery of NO, there isidentical concentration dependent ascorbate enhancement of inhibitionwith the 2.5 mM NO-GSH and NO-LB+2.5 mM GSH.

FIGS. 10A-B. Influences of bicarbonate on alginate production bydifferent strains of P. aeruginosa under a 5% CO₂ atmosphere. Alginateproduction of selected strains of P. aeruginosa surface grown in a CO₂incubator was quantitatively determined by a colorimetric assay.Previous studies suggested that clinical isolates of mucoidy P.aeruginosa had exaggerated mucoidy production when grown in 5% CO₂atmosphere (A, left plate) vs. ambient atmosphere (A, right plate). Incontrast, strain PAO1 does not produce mucoidy colonies, but Pa Mucoidlaboratory derived from PAO1 does. Colonies from Pa Mucoid however werenot affected by 5% CO₂ compared to ambient atmosphere. These threestrains were then tested for their ability to make alginate under anatmosphere of 5% CO₂ with increasing concentrations of NaHCO₃ (B). Ascan be seen, PAO1 failed to make alginate under any of the testconditions. The clinical isolate (C3873M) made significant quantities ofalginate and this alginate production was inhibited by bicarbonate. Incontrast alginate production by the laboratory strain not only was notsuppressed, but was significantly enhanced by bicarbonate. These datasuggest that alginate production provides an important barrier toreactive oxygen species that would be associated with the exaggeratedinflammatory response in the CF airway. These data also suggest thatmucoidy properties are important to bacterial tolerance of the RNIassociated with nitrate respiration. This would indicate that raisingthe levels of bicarbonate in the bicarbonate-deficient airway surfaceliquid of the CF lung could shut down an important defense mechanism ofCF pathogens.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will now be described more fully hereinafter. Thisinvention may, however, be embodied in different forms and should not beconstrued as limited to the embodiments set forth herein. Rather, theseembodiments are provided so that this disclosure will be thorough andcomplete, and will fully convey the scope of the invention to thoseskilled in the art.

The terminology used in the description of the invention herein is forthe purpose of describing particular embodiments only and is notintended to be limiting of the invention.

Unless otherwise defined, all terms (including technical and scientificterms) used herein have the same meaning as commonly understood by oneof ordinary skill in the art to which this invention belongs. It will befurther understood that terms, such as those defined in commonly useddictionaries, should be interpreted as having a meaning that isconsistent with their meaning in the context of the present applicationand relevant art and should not be interpreted in an idealized or overlyformal sense unless expressly so defined herein. The terminology used inthe description of the invention herein is for the purpose of describingparticular embodiments only and is not intended to be limiting of theinvention. All publications, patent applications, patents and otherreferences mentioned herein are incorporated by reference in theirentirety.

A subject “in need thereof” as used herein refers to a subject that canbenefit from the therapeutic and/or prophylactic effects of thepharmaceutical compositions of the present invention. Such a subject canbe a subject diagnosed with a disease or disorder of this invention, asubject suspected of having or developing a disorder or disease of thisinvention, and/or a subject determined to be at increased risk of havingor developing a disease or disorder of this invention.

By the term “treat,” “treating,” or “treatment of” (and grammaticalvariations thereof) it is meant that the severity of the subject'scondition is reduced, at least partially improved or ameliorated, and/orthat some alleviation, mitigation or decrease in at least one clinicalsymptom is achieved and/or there is a delay in the progression of thedisease or disorder.

The terms “prevent,” “preventing,” and “prevention of” (and grammaticalvariations thereof) refer to reduction and/or delay of the onset and/orprogression of a disease, disorder and/or a clinical symptom(s) in asubject and/or a reduction in the severity of the onset and/orprogression of the disease, disorder and/or clinical symptom(s) relativeto what would occur in the absence of the methods of the invention. Theprevention can be complete, e.g., the total absence of the disease,disorder and/or clinical symptom(s). The prevention can also be partial,such that the occurrence of the disease, disorder and/or clinicalsymptom(s) in the subject and/or the severity of onset and/or theprogression is less than what would occur in the absence of acomposition of the present invention.

As used herein, the terms “therapeutically effective amount” or“effective amount” refer to an amount of a composition or formulation ofthis invention that elicits a therapeutically useful response in asubject. Those skilled in the art will appreciate that the therapeuticeffects need not be complete or curative, as long as some benefit isprovided to the subject.

As used in the description of the invention and the appended claims, thesingular forms “a,” “an” and “the” are intended to include the pluralforms as well, unless the context clearly indicates otherwise. Forexample, “a cell” can mean a single cell or a multiplicity of cells.

Also as used herein, “and/or” refers to and encompasses any and allpossible combinations of one or more of the associated listed items, aswell as the lack of combinations when interpreted in the alternative(“or”).

As used herein, the transitional phrase “consisting essentially of” (andgrammatical variants) is to be interpreted as encompassing the recitedmaterials or steps “and those that do not materially affect the basicand novel characteristic(s)” of the claimed invention. See, In re Herz,537 F.2d 549, 551-52, 190 U.S.P.Q. 461, 463 (CCPA 1976) (emphasis in theoriginal); see also MPEP §2111.03. Thus, the term “consistingessentially of” as used herein should not be interpreted as equivalentto “comprising.”

The term “about,” as used herein when referring to a measurable valuesuch as an amount or concentration (e.g., the amount of thebenzodiazepine in the pharmaceutical composition) and the like, is meantto encompass variations of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% of thespecified amount.

Unless the context indicates otherwise, it is specifically intended thatthe various features of the invention described herein can be used inany combination. For example, features described in relation to oneembodiment may also be applicable to and combinable with otherembodiments and aspects of the invention.

Moreover, the present invention also contemplates that in someembodiments of the invention, any feature or combination of features setforth herein can be excluded or omitted. To illustrate, if thespecification states that a complex comprises components A, B and C, itis specifically intended that any of A, B or C, or a combinationthereof, can be omitted and disclaimed.

Pharmaceutical Compositions

The present invention is based on the discovery that pharmaceuticalcompositions of this invention, comprising for example in someembodiments, glutathione, an organic acid (e.g., ascorbic acid),bicarbonate and, optionally, thiocyanate can be used as an antimicrobialand/or anti-inflammatory agent to treat diseases and disorders inmucosal tissue, as well as to establish, reestablish, and/or maintainthe homeostatic environment in mucosal surface fluid to restore orenhance the subject's natural healing and defense mechanisms. The reasonglutathione hasn't worked in the past is that it must also have theactivities of bicarbonate and ascorbate as well to bring in to playother complementary innate defenses and anti-inflammatory molecules.Likewise, the addition of thiocyanate arms another component of exocrinesecretions with both antimicrobial and anti-inflammatory activities. Itis not simply the buffering capacity of bicarbonate that is important,the function of lactoferrin has specificity in its coordinate binding ofiron for carbonate/bicarbonate and their ratio is central to determiningboth the antimicrobial and anti-inflammatory functions of this criticalexocrine protein. Likewise, ascorbate was chosen not simply because itis an organic acid, but because of its iron reducing potential and itsspecific and unique relationship to the functions of lactoferrin.Carbonate provokes exaggerated expression of the mucoidy trait(associated with virulence) of Pseudomonas aeruginosa and this issuppressed specifically by bicarbonate, presumably facilitating hostdefense against this critical airway pathogen. The uniqueimportance/functions of ascorbate and bicarbonate, both of which aredeficient in the cystic fibrosis (CF) airway and in other secretionsthat bathe mucosal surfaces, in addition to glutathione with or withoutthiocyanate addresses previous shortcomings in the art.

Thus, the present invention provides various compositions andformulations as set forth herein.

A composition of this invention can comprise, consist essentially of orconsist of a) glutathione, a pharmaceutically-acceptable salt ofglutathione, or a derivative or a prodrug thereof, b) an organic acid, apharmaceutically-acceptable salt of an organic acid, such as ascorbicacid, or a derivative or prodrug thereof, and c) a bicarbonate salt,such as sodium or potassium bicarbonate. A pH adjusting agent can alsobe present in the composition to adjust the pH of the composition tofall within a range from about 5 to about 9, from about 6 and about 8,or from about 6.5 to about 7.5.

The weight ratios of these components (i.e., active agents) can varyover wide ranges. Typically, the amount of each component is selectedsuch that there is sufficient bicarbonate salt to bring the pH to thedesired range, while also allowing for the bicarbonate salt to bepresent along with the glutathione and organic acid.

It is to be understood that, as the relative amounts of glutathione andorganic acid, such as ascorbic acid, can vary, the amounts ofbicarbonate salt can also vary. If glutathione and the organic acid aremixed with a bicarbonate salt, the acid functional groups will reactwith the bicarbonate to form the salts of the glutathione and theorganic acid, and the bicarbonate will be acidified to form carbonicacid, with concomitant formation of water and evolution of carbondioxide. Accordingly, if the formulation is prepared by mixing the acidswith a bicarbonate salt, what is intended is that sufficient bicarbonateis added wherein the bicarbonate salt is present in a molar amount equalto the combined molar amount of the glutathione and the organic acid orin a molar excess of the combined molar amount of the glutathione andthe organic acid. For example, the molar excess of the bicarbonate saltcan be in a range from greater than 1.0 to less than about 1.5.

The weight ratios of these components can vary over wide ranges. Forexample, the weight of a) can be from about 0.1 to about 95 percent byweight, b) can be from about 0.5 to about 60 percent by weight, and c)can be from about 1 to about 55 percent by weight. Within these ranges,the amount of a) can be, for example, from about 52 to about 80 weightpercent, b) can be from about 3 to about 18 weight percent, and c) canbe from about 17 to about 30 weight percent.

In some embodiments, glutathione precursors such as methionine, cysteineor N-acetyl cysteine (NAC) can be included in the compositions of thisinvention in addition to, or in place of, glutathione. These precursorsenable glutathione to be produced intracellularly. This can be importantin certain embodiments, as glutathione is not taken up by cells, andwill not work if supplied on the outside of the cell. So, for example,for perfusion solutions to preserve transplant organs between harvestand transplantation, it can be advantageous to use a glutathioneprecursor in a composition of this invention. In some embodiments, acomposition of this invention comprising a glutathione precursor inplace of glutathione can be used to perfuse the vasculature of an organand a composition of this invention comprising glutathione can be usedto perfuse the airway of an organ, to preserve endothelial function.Thus, the compositions and formulations of this invention can be used topreserve both mucosal and endothelial tissue.

In some embodiments, a composition of this invention can furthercomprise, consist essentially of or consist of (e.g., as an additionalactive agent) from about 0.01% to about 5% by weight of apharmaceutically-acceptable thiocyanate salt, such as from about 1% toabout 2% by weight of a thiocyanate salt, while maintaining the weightratios of the other components.

In some embodiments, the compositions can further include precursors toglutathione, such as methionine, cysteine and/or N-acetyl cysteine, inamounts of from about 0.1 to about 10% by weight, while maintaining theweight ratios of the other components. In one aspect of this embodiment,a glutathione precursor such as methionine, cysteine and/or N-acetylcysteine can be used in place of glutathione, rather than in addition toglutathione. In this aspect, the dosage of the glutathione precursor canbe that of glutathione.

The pharmaceutical compositions described herein can be present in theform of solid formulations, such as particulate formulations, or insolution form. When in particulate form, the particles can be mixed withgases, or liquid propellants, for use in inhalation therapy. Other solidformulations include formulations for oral administration, but buccaladministration or colonic administration, and suppositories for rectalor vaginal administration. Representative formulations include, but arenot limited to, the following: eye drops, nebulizers, topical gels andointments, dry powders, particles, sprays, liquids, anesthetic machinesor vaporizers, autoinjectors, intrauterine devices, respimats,liniments, liposomes, lotions, formulations for intramuscular,intrathecal, or subcutaneous injection, douches, infusions, and facemasks.

In solution form, the formulations can be in the form of sprays forintranasal administration, formulations for use in nebulizers, andformulations for rectal administration, such as enemas and colonies.Solutions that include water-miscible organic solvents, such aspropylene glycol and/or glycerol, and other components normally found invaginal and rectal lubricants, can also be used. Regardless of thesolvents used, the solvent is typically present in a weight ratio offrom about 15 to about 85 percent by weight, relative to the weight ofthe solids, and, more typically, is from about 50 to about 85% byweight.

In some embodiments, the present invention provides a pharmaceuticalcomposition comprising glutathione, a pharmaceutically acceptable saltthereof, a derivative thereof, an analogue thereof and/or a prodrugthereof, an organic acid, a pharmaceutically acceptable salt thereof, aderivative thereof, an analogue thereof and/or a prodrug thereof, and abicarbonate. In some embodiments, this pharmaceutical composition canfurther comprise thiocyanate, a pharmaceutically acceptable saltthereof, a derivative thereof, an analogue thereof and/or a prodrugthereof.

In some embodiments, the pharmaceutical composition of this inventioncan comprise glutathione that is oxidized glutathione.

In some embodiments, the pharmaceutical composition of this inventioncan comprise glutathione that is reduced glutathione.

In various embodiments of this invention, the liquid pharmaceuticalcompositions of this invention can have a pH in a range from about 2.0to about 10 (e.g., 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5,7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0). In some embodiments, the pH can bein a range from about 5.5 to about 9.0).

A pharmaceutical composition of this invention can further comprise achelating agent and/or a preservative.

The pharmaceutical composition of this invention can be formulated forcontrolled release (i.e., in a controlled release formulation).

In some embodiments, the pharmaceutical composition of this inventioncan be in a form for administration by inhalation, as described hereinand as is well known in the art.

In some embodiments, the pharmaceutical composition of this inventioncan be in a form for intranasal administration, as described herein andas is well known in the art.

In some embodiments, the pharmaceutical composition of this inventioncan be in a form for administration to an eye, as described herein andas is well known in the art.

In some embodiments, the pharmaceutical composition of this inventioncan be in a form for administration to an ear, as described herein andas is well known in the art.

In further embodiments, the pharmaceutical composition of this inventioncan be in a form for administration to a mucosal surface, as describedherein and as is well known in the art. Nonlimiting examples of tissueswith mucosal surface/lining include the mouth, nose, eye, ear, upperrespiratory tract, lower respiratory tract, gastrointestinal tract,vagina, rectum and urethra.

Glutathione and Derivatives Thereof

The term “glutathione” encompasses the tripeptideN—(N-L-γ-glutamyl-L-cysteinyl)glycine, often called L-glutathione,glutathione-SH, or γ-Glu-Cys-Gly, and sold under a variety of tradenamessuch as Agifutol S™, Copren™, Deltathione™, Isethion™, Neuthion™,Tathiclon™, Tathion™ and Triptide™. As used herein, where the propertiesand advantages of “glutathione” (or GSH) are discussed as an activeingredient in the practice of the invention, biologically activeglutathione derivatives are encompassed. “Glutathione derivatives”include, but are not limited to, reduced glutathione (or GSSG),glutathione salts, particularly reduced glutathione potassium or sodiumsalts.

In some embodiments, glutathione derivatives of this invention caninclude, but are not limited to, glutathione prodrugs, including but notlimited to glutathione alkyl esters, particularly C₁ to C₁₀ alkylesters, especially monoesters such as monomethyl and monoethyl esters,which have the glycine carboxylic acid group acylated, as these havebeen shown to increase cellular levels of glutathione (U.S. Pat. No.4,710,489 to Meister), and corresponding amides and imides (such asthose set out in U.S. Pat. No. 5,541,162 to Ohmori, et al).

In other embodiments, glutathione derivatives can include, but are notlimited to, glutathione prodrugs, including but not limited tosulfhydryl-modified prodrugs of glutathione (Berkeley et al.“Hepatoprotection by L-cysteine-glutathione mixed disulfide, asulfhydryl-modified prodrug of glutathione” J. Biochem. Mol. Toxicol.17:95-97 (2003)) diesters based onN-benzyloxycarbonyl-S-2,4-dinitrophenylglutathione (Daunes et al.“Glutathione Derivatives Active against Trypanosoma brucei rhodesienseand T. brucei brucei In Vitro” Antimicrobial Agents and Chemother.46(2):434-437 (2002)) and glutathione prodrugs (Cacciatore et al.“Prodrug Approach for Increasing Cellular Glutathione Levels” Molecules15:1242-1264 (2010)).

In other embodiments, glutathione derivatives of this invention caninclude, but are not limited to, analogues (Cacciatore et al. “Synthesisand activity of novel glutathione analogues containing an urethanebackbone linkage” Farmaco 58(9):787-793 (2003)), isosteres (Cacciatoreet al. “Transition State Isosteres of the γ-Glutamyl Peptide BondHydrolysis: Synthesis and Characterization of the ψ (CH2NH)Pseudopeptide Analogue of Glutathione” J. Peptide Sci. 10:109-114(2004)) and conformationally restricted glutathione analogues(Paglialunga et al, “Proline-glutamate chimeras in isopeptides,Synthesis and biological evaluation of conformationally restrictedglutathione analogues” Bioorg. Med. Chem. 11:1677-1683 (2003)).

In other embodiments, configurational isomers, optically pureenantiomers, mixtures of enantiomers such as racemates, diastereomers,mixtures of diastereomers, diastereomeric racemates, mixtures ofdiastereomeric racemates and the meso-form as well as pharmaceuticallyacceptable salts of glutathione and glutathione derivatives areincluded. The present invention encompasses all these forms.

In some embodiments, glutathione precursors, such as methionine,cysteine or NAC are used, in addition to, or in place of glutathione.The dosage can mirror that of the glutathione.

The present invention furthermore encompasses glutathione andderivatives of glutathione, for example solvates, such as hydrates andadducts with alcohols, esters, prodrugs and other physiologicallytolerated derivatives and also active metabolites of glutathione.Furthermore, the invention contains all crystal modifications ofglutathione and glutathione derivatives.

In still other embodiments, some of the crystalline forms of glutathioneand the glutathione derivatives may exist as polymorphs, which areincluded in the present invention.

Organic Acids

In the pharmaceutical compositions recited above, the organic acid canbe, but is not limited to, ascorbic, acetic, adipic, aspartic,benzenesulfonic, benzoic, butyric, camphorsulfonic, camsylic, carbonic,chlorobenzoic, cholic, citric, edetic, edisylic, estolic,ethanesulfonic, formic, fumaric, gluceptic, gluconic, glucuronic,glutamic, glycolic, glycolylarsanilic, hippuric, 1-hydroxy-2-naphthoic,isethionic, isobutyric, isonicotinic, lactic, lactobionic, maleic,malic, malonic, mandelic, methanesulfonic, mucic, muconic,napthalenesulfonic, nicotinic, oxalic, oleic, orotic,p-nitromethanesulfonic, pamoic, pantothenic, phthalic,polygalactouronic, propionic, saccharic, salicylic, stearic, suberic,succinic, sulfanilic, tannic, tartaric, p-toluenesulfonic and anycombination thereof.

pH Adjusting Agents

Suitable pH adjusting agents are well-known in the art (see Remington'sPharmaceutical Sciences, 18th edition, A. R Gennaro, Ed., MackPublishing Company (1990) and Handbook of Pharmaceutical Excipients, 3rdedition, A. Kibbe, Ed., Pharmaceutical Press (2000). Suitable examplesof pharmaceutically acceptable pH adjusting agents include, but are notlimited to, citric acid, sodium citrate, sodium bicarbonate, dibasicsodium phosphate, magnesium oxide, calcium carbonate and magnesiumhydroxide. Suitable pH adjusting agents for use with the inventioninclude, but are not limited to, acetate buffers, citrate buffers,phosphate buffers, lactic acid buffers, and borate buffers.

Fat-soluble fatty acid esters of ascorbic acid (vitamin C) are employedas an adjunct ingredient in some embodiments, alone or in combinationwith α-hydroxy acids. The more oxidation-resistant saturated fatty acidesters of ascorbic acid can be used, including, but not limited to,ascorbyl laurate, ascorbyl myristate, ascorbyl palmitate, ascorbylstearate, and ascorbyl behenate. Ascorbyl palmitate can be used in someembodiments. As denoted herein, where fatty acid esters are described,e.g., ascorbyl stearate, compositions having predominantly that ester,e.g., predominantly stearate, are included. The esters may be preparedusing hydrogenated oils or fats, or fractions thereof, and contain smallamounts of another ester. Ascorbyl stearate prepared using canola, forexample, can commonly contain about 4% ascorbyl palmitate.

Preservatives

Suitable examples of pharmaceutically acceptable preservatives include,but are not limited to, various antibacterial and antifungal agents,solvents, for example, ethanol, propylene glycol, benzyl alcohol andchlorobutanol, quaternary ammonium salts including, but not limited to,cetylypridinium chloride, benzalkonium chloride and parabens including,but not limited to, methyl paraben, ethyl paraben and propyl paraben.

In other embodiments, pharmaceutically acceptable preservatives of thisinvention can include, but are not limited to, chlorhexidine, benzoicacid and the salts thereof, parahydroxybenzoic acids and the saltsthereof, alkyl esters of parahydroxybenzoic acid and the salts thereof,phenylmercuric salts such as nitrate, chloride, acetate, and borate, orantioxidants, as well as additives like EDTA, sorbitol and the like.

In still other embodiments, pharmaceutically acceptable preservatives ofthis invention can include, but are not limited to, phenol, boric acidand the salts thereof, sorbic acid and the salts thereof, thimerosal andnitromersol.

Chelating Agents

Non-limiting examples of chelating agents suitable for use in thepresent invention include lactic acid, acetic acid, oxalic acid, malonicacid, succinic acid, maleic acid, fumaric acid, aconitic acid, pimelicacid, sebacic acid, allymalonic acid, ethylmalonic acid, citric acid,malic acid, glyceric acid, tartaric acid, mevaloic acid, oxyglutaricacid, oxaloacetic acid, α-ketoglutaric acid, β-ketoglutaric acid,α-ketomalonic acid, glucuronic acid, galaceturonic acid, mannuronicacid, aspartic acid, glutamic acid, glycine, alanine, lysine, histidine,alginine, cysteine, ε-aminocaproic acid, phenylalanine, phenylglycine,p-hydroxyphenylglycine, p-aminophenylalanine, γ-carboxyglutamic acid,iminodiacetic acid, hydroxyethyliminodiacetic acid,ethylenediaminediacetic acid, ethylenediaminetetraacetic acid,trans-cyclohexane-diaminetetraacetic acid, diethylenediaminepentaaceticacid, β-alaninediacetic acid, diaminopimelic acid, phthalic acid,terephthalic acid, homophthalic acid, phenylsuccinic acid, phenylmalonicacid, oxanylic acid-o-carboxylic acid, anthralininoacetic acid,2,4-dihydroxybenzoic acid, p-aminosalicyclic acid, phthalyglutamic acid,kynurenine, 1,2-hyroxybenzene-3,5-disulfonic acid,4-amino-phenol-2-sulfonic acid, cysteic acid, 2-phosphoglyceric acid,glycero-3-phosphoric acid, glucose-1,6-diphosphoric acid,fructose-1,6-diphosphoric acid and phosphates (e.g., sodium phosphate,sodium aluminum phosphate, sodium acid phosphate, dipotassium phosphate,disodium phosphate, monobasic and sodium hexametaphosphate) and anycombination thereof.

Chelating agents can be included in the pharmaceutical compositions ofthis invention either as the parent molecule or in the salt form whereappropriate. For example, compounds containing an acid function may beused in the protonated form or as a pharmaceutically acceptableinorganic or organic salt which retains the chelating activity of theparent compound.

Pharmaceutically-Acceptable Salts

The pharmaceutically-acceptable salts will be any salt that is notdeleterious to the subject or otherwise contraindicated. Such salts arefor example, salts derived from inorganic bases which include sodium,potassium, lithium, ammonium, calcium, magnesium, ferrous, zinc, copper,manganous, aluminum, ferric, manganic salts and the like. Particularsalts include the ammonium, potassium, sodium, calcium and magnesiumsalts. Salts derived from pharmaceutically acceptable organic non-toxicbases include salts of primary, secondary, and tertiary amines,substituted amines including naturally occurring substituted amines,cyclic amines and basic ion exchange resins, such as isopropylamine,trimethylamine, diethylamine, triethylamine, tripropylamine,ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol,tromethamine, dicyclohexylamine, lysine, arginine, histidine, caffeine,procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine,methylglucamine, theobromine, purines, piperazine, piperidine,N-ethylpiperidine, polyamine resins and the like. Organic non-toxicbases can be isopropylamine, diethylamine, ethanolamine, tromethamine,dicyclohexylamine, choline and caffeine.

Where the chelating agent has more than one acid function, one or moreof the acid functions may be in the salt form. For example, EDTA may beused as the mono, di, tri or tetrasodium salt or the disodium,monocalcium salt may be used as an alternative. Particular examples of achelating agent of this invention include the alkali metal salts ofEDTA. Further examples include the mono and disodium salt of EDTA.

Therapeutic Agents

In addition to the components of the formulations described above, theformulations can also include one or more therapeutic agents.

In still further embodiments, the composition is combined with one ormore additional therapeutic agents. Representative therapeutic agentsinclude, but are not limited to, monoclonal antibodies, immunomodulatoryagents, including agents that cause T and B cell activation,proliferation, and/or maturation; agents that bring about innate immunesystem activation, proliferation, maturation (e.g., JNK, MAPK, ERK, NKkappa B pathway agonists or antagonists, and monocyte, neutrophil, ormacrophage agonists or antagonists), matrix metalloproteinaseinhibitors, heat shock protein agonists or antagonists, alpha synucleininhibitors, chelating agents, diuretics, alpha 1 antitrypsin modulators,purinoceptor agonists or antagonists, cyclooxygenase 2 inhibitors, DNAgyrase inhibitors, natural killer cell and natural killer T cellagonists or antagonists, cathepsin class agonists or antagonists,antioxidant therapies, rho-associated kinase inhibitors, myosininhibitors, phosphatidylinositol 3 kinase inhibitors and relatedmolecules, nitric oxide synthase agonists or antagonists, nitric oxideagonists or antagonists, ion channel function or trafficking modulators,surfactant therapies, in particular, lung surfactants, cannabinoidreceptor modulators, complement system inhibitors, IgE receptorantagonists, G protein-coupled receptor agonists or antagonists,chemokines, chemokine receptor agonists or antagonists, cytokines, andcytokine receptor agonists or antagonists, arachidonic acid agonists orantagonists, inflammatory mediators, STAT6 inhibitors, histamine orleukotriene agonists or antagonists, and calcineurin agonists orantagonists. Examples of these therapeutic agents and their use aredescribed in more detail below.

Matrix Metalloproteinase Inhibitors

Matrix metalloproteinase inhibitors (MMPIs) inhibit matrixmetalloproteinases, and have antiangiogenic effects. MMPIs can beendogenous or exogenous. Examples of endogenous metalloproteinasesinclude tissue inhibitors of metalloproteinases (TIMPs) andcartilage-derived angiogenesis inhibitors. Exogenous matrixmetalloproteinase inhibitors that were developed as anticancer drugsinclude Batimastat and Marimastat.

All cancerous tumors release angiogenic growth factor proteins thatstimulate blood vessels to grow into the tumor, providing it with oxygenand nutrients. Antiangiogenic therapies starve the tumor of its bloodsupply. Cancer treatments that block angiogenesis are now approved andavailable to treat cancers of the colon, kidney, lung, breast, liver,brain, and thyroid, as well as multiple myeloma, bone gastrointestinalstromal tumors, soft tissue sarcoma, and SEGA tumors. Otherangiogenesis-dependent conditions include hemangiomas, colon polyps, andprecancerous skin lesions. MMP enzymes are also thought to contribute tothe deterioration of cartilage in osteoarthritis patients.

MMPIs can therefore be used for their antiangiogenic properties, in thetreatment of cancer, arthritis, and other disorders associated withangiogenesis.

When treating certain forms of lung cancer, it can be helpful tomaintain the homeostasis of the lung tissue, so co-administration orseparate administration of chemotherapeutic agents such as MMPIs and theglutathione/ascorbic acid/sodium bicarbonate formulations describedherein can be used for this purpose. That is, the MMPIs can be used fortheir antiangiogenic properties, as well as for their anti-inflammatoryproperties, to treat cancer, arthritis, and other disorders.Co-administration or separate administration of chemotherapeutic agentssuch as MMPIs and the glutathione/ascorbic acid/sodium bicarbonateformulations described herein can be used to maintain homeostasis in themucosal membranes of the lung tissue.

Monoclonal Antibodies

Monoclonal antibodies (mAb or moAb) are monospecific antibodies producedby identical immune cells that are all clones of a unique parent cell,in contrast to polyclonal antibodies which are made from severaldifferent immune cells. Monoclonal antibodies have monovalent affinity,in that they bind to the same epitope.

Monoclonal antibody therapy is the use of monoclonal antibodies (or mAb)to specifically bind to target cells or proteins. This may thenstimulate the patient's immune system to attack those cells. It ispossible to create a mAb specific to almost any extracellular/cellsurface target, and thus there is a large amount of research anddevelopment currently being undertaken to create monoclonals fornumerous serious diseases (e.g., rheumatoid arthritis, multiplesclerosis, Alzheimer's disease and different types of cancers). Thereare a number of ways that mAbs can be used for therapy. For example: mAbtherapy can be used to destroy malignant tumor cells and prevent tumorgrowth by blocking specific cell receptors. Variations also exist withinthis treatment (e.g. radioimmunotherapy) where a radioactive doselocalizes on target cell line, delivering lethal chemical doses to thetarget. Examples of monoclonal antibodies used include, but are notlimited to, Adalimumab, Alemtuzumab, Basiliximab and Omalizumab.

Monoclonal antibody therapies are used for a variety of diseases, suchas asthma autoimmune diseases, cancers, and general immunosuppression.When the therapeutic agents described herein are used in combinationwith the antibodies, one can reduce dosages of monoclonal antibodytherapies, thereby limiting exposure to toxic drug levels. Additionally,the compounds can be used in conjunction with monoclonal antibodiesand/or other therapies to increase efficacy and/or decrease sideeffects.

Monoclonal antibodies that bind only to cancer cell-specific antigensand induce an immunological response against the target cancer cell canbe used to treat cancer. Such mAbs can be modified for delivery of atoxin, radioisotope, cytokine or other active conjugate. MAbs currentlyapproved by the U.S. Food and Drug Administration (USFDA) includeBevacizumab, Cetuximab, Panitumumab and Trastuzumab.

Monoclonal antibodies used for autoimmune diseases include infliximaband adalimumab, which are effective in rheumatoid arthritis, Crohn'sdisease and ulcerative Colitis by their ability to bind to and inhibitTNF-α. Basiliximab and daclizumab inhibit IL-2 on activated T cells andthereby help prevent acute rejection of kidney transplants. Omalizumabinhibits human immunoglobulin E (IgE) and is useful inmoderate-to-severe allergic asthma.

Immunomodulatory Agents

Immunotherapy is a medical term defined as the “treatment of disease byinducing, enhancing, or suppressing an immune response.” Immunotherapiesdesigned to elicit or amplify an immune response are classified asactivation immunotherapies, while immunotherapies that reduce orsuppress are classified as suppression immunotherapies.

The therapeutic agents of immunotherapy are collectively calledimmunomodulators. They are a diverse array of recombinant, synthetic andnatural preparations, often cytokines. Some of these substances, such asgranulocyte colony-stimulating factor (G-CSF), interferons, imiquimodand cellular membrane fractions from bacteria are already licensed foruse in patients. Others including IL-2, IL-7, IL-12, various chemokines,synthetic cytosine phosphate-guanosine (CpG), oligodeoxynucleotides andglucans are currently being investigated extensively in clinical andpreclinical studies. Immunomodulatory regimens offer an attractiveapproach as they often have fewer side effects than existing drugs,including less potential for creating resistance in microbial diseases.

Cell-based immunotherapies have proven to be effective for some cancers.Immune effector cells such as lymphocytes, macrophages, dendritic cells,natural killer cells (NK cell), cytotoxic T lymphocytes (CTL), etc.,work together to defend the body against cancer by targeting abnormalantigens expressed on the surface of the tumor due to mutation.

Immune suppression dampens an abnormal immune response in autoimmunediseases or reduces a normal immune response to prevent rejection oftransplanted organs or cells.

Immunosuppressive drugs are important tools in the management of organtransplantation and autoimmune disease. Immune responses depend onlymphocyte proliferation, and cytostatic drugs are immunosuppressive.Glucocorticoids are somewhat more specific inhibitors of lymphocyteactivation, whereas inhibitors of immunophilins more specifically targetT lymphocyte activation. Immunosuppressive antibodies target anincreasingly-broad array of steps in the immune response, and there arestill other drugs that modulate immune responses.

Immune tolerance is the process by which the body naturally does notlaunch an immune system attack on its own tissues. Immune tolerancetherapies seek to reset the immune system so that the body stopsmistakenly attacking its own organs or cells in autoimmune disease oraccepts foreign tissue in organ transplantation. A brief treatmentshould then reduce or eliminate the need for lifelong immunosuppressionand the chances of attendant side effects, in the case oftransplantation, or preserve the body's own function, at least in part,in cases of type 1 diabetes or other autoimmune disorders. Anotherexample of this is using helminthic therapies to modulate the immunesystem (e.g., Crohn's disease).

A potential use of immunotherapy is to restore the immune system ofpatients with immune deficiencies as result of infection orchemotherapy. For example, cytokines have been tested in clinicaltrials; interleukin-7 has been in clinical trials for HIV and cancerpatients. In addition, interleukin-2 has also been tested in HIVpatients.

Anti-microbial immunotherapy, which includes vaccination, involvesactivating the immune system to respond to an infectious agent.

Examples of immunomodulatory agents include, but are not limited to,tacrolimus, mycophenolate sodium, corticosteroids, innate immune systemactivation, proliferation, maturation (e.g., JNK (Jun amino-terminalkinases), MAPK (Mitogen-activated protein kinase), ERK (extracellularsignal-regulated kinases), NK kappa B (nuclear factorkappa-light-chain-enhancer of activated B cells) pathway agonists orantagonists, and monocyte, neutrophil, or macrophage agonists orantagonists), STAT6 inhibitors, natural killer cell and natural killer Tcell agonists or antagonists, calcineurin agonists or antagonists(partial agonists, inverse agonists, and allosteric modulators),chemokines, chemokine receptor agonists or antagonists, cytokines, andcytokine receptor agonists or antagonists.

Immunomodulators weaken or modulate the activity of the immune system,which, in turn, decreases the inflammatory response. Immunomodulatorsare most often used in organ transplantation to prevent rejection of thenew organ, in autoimmune diseases such as rheumatoid arthritis, and topatients with irritable bowel disorder (IBD), which appears to be causedby an overactive immune system. Immunomodulators are often combined withcorticosteroids to speed up response during active flares of disease.

Representative immunomodulatory agents also include agents that cause Tand B cell activation, proliferation, and/or maturation; agents thatbring about innate immune system activation, proliferation, maturation

Representative agents which modulate NK kappa B include denosumab,Disulfuram, olmesartan, dithiocarbamates, and Anatabine.

Heat Shock Protein Agonists/Antagonists/Partial Agonists/InverseAgonists

Heat shock proteins (HSP) are a group of proteins induced by heat shock.The most prominent members of this group are a class of functionallyrelated proteins involved in the folding and unfolding of otherproteins. Their expression is increased when cells are exposed toelevated temperatures or other stress.

Heat Shock Factor 1 (HSF1) is a transcription factor that is involved inthe upregulation of Hsp70 protein expression. Recently it was discoveredthat HSF1 is a powerful multifaceted modifier of carcinogenesis.

Given their role in antigen presentation, HSPs are useful as immunologicadjuvants in boosting the response to a vaccine. Furthermore, someresearchers speculate that HSPs may be involved in binding proteinfragments from dead malignant cells and presenting them to the immunesystem. Therefore HSPs may be useful for increasing the effectiveness ofcancer vaccines.

Intracellular heat shock proteins are highly expressed in cancerouscells and are essential to the survival of these cell types. Hence smallmolecule inhibitors of HSPs, especially Hsp90 show promise as anticanceragents. The potent Hsp90 inhibitor 17-AAG is currently in clinicaltrials for the treatment of several types of cancer. HSPgp96 also showspromise as an anticancer treatment and is currently in clinical trialsagainst non-small cell lung cancer.

Alpha Synuclein Inhibitors

Alpha-synuclein is a protein that, in humans, is encoded by the SNCAgene. An alpha-synuclein fragment, known as the non-Abeta component(NAC) of Alzheimer's disease amyloid, originally found in anamyloid-enriched fraction, was shown to be a fragment of its precursorprotein, NACP. NACP is referred to as human alpha-synuclein.

Alpha synuclein defects and/or deficiencies are implicated in CNSdisorders, such as Alzheimer's and Parkinson's diseases. Congenitaldefect or otherwise depletion of this protein is associated withincreased oxidative stress and inflammation in the brain, which has beenshown to directly contribute to the onset and progression of Alzheimer'sand Parkinson's diseases, and other degenerative CNS disorders. Theoxidative stress and inflammation lead to a depletion of glutathione andascorbate in neurons and supporting cells. Therefore this therapy hasthe potential to reduce dosages of therapies associated withmanipulating alpha-synuclein function or expression, thereby limitingexposure to toxic drug levels. Additionally, it can be used inconjunction with alpha-synuclein and other therapies to increaseefficacy and/or decrease side effects.

Representative inhibitors include Posiphen and certain flavonoids.

Chelating Agents

Chelation describes a particular way that ions and molecules bind metalions, namely, the formation or presence of two or more separatecoordinate bonds between a polydentate (multiple bonded) ligand and asingle central atom. Usually these ligands are organic compounds.

Chelating agents are used in chelation therapy to detoxify poisonousmetal agents such as mercury, arsenic, and lead by converting them to achemically inert form that can be excreted without further interactionwith the body. In alternative medicine, chelation is used as a treatmentfor autism.

EDTA is also used in root canal treatment as an intracanal irrigant.EDTA softens the dentin which may improve access to the entire canallength and is used as an irrigant to assist in the removal of the smearlayer.

Chelate complexes of gadolinium are often used as contrast agents in MRIscans.

Diuretics

Diuretics promote urine production, increasing water excretion.Antidiuretics such as vasopressin reduce water excretion in urine.Diuretics are used to treat heart failure, liver cirrhosis, hypertensionand certain kidney diseases. Some diuretics, such as acetazolamide, helpto make the urine more alkaline and are helpful in increasing excretionof substances such as aspirin in cases of overdose or poisoning.

Diuretics include high ceiling loop diuretics, such as furosemide,ethacrynic acid, torsemide, and furosemide; thiazides, carbonicanhydrase inhibitors such as acetazolamide and methazolamide,potassium-sparing diuretics, aldosterone antagonists, such asspironolactone, eplerenone, and potassium canreonate; epithelial sodiumchannel blockers such as amiloride and triamterene, calcium-sparingdiuretics (thiazides are an example of such diuretics); osmoticdiuretics, including glucose and mannitol, and low ceiling diuretics.

Alpha 1 Antitrypsin Modulators

Alpha-1 Antitrypsin or α1-antitrypsin (A1AT) is a protease inhibitorwhich belongs to the serpin superfamily and is generally known as serumtrypsin inhibitor. Alpha 1-antitrypsin is also referred to as alpha-1proteinase inhibitor (A1PI) because it inhibits a wide variety ofproteases. It protects tissues from enzymes of inflammatory cells,especially neutrophil elastase

Recombinant A1PI include Prolastin, Zemaira, and Aralast.

Trypsin inhibitors include oxidoreductases, including Aldose reductase•HMG-CoA reductase, 5-alpha-reductase, Monoamine oxidase, Dihydrofolatereductase, Lipoxygenase, Aromatase, COX-2, Xanthine oxidase, andRibonucleotide reductase; Transferases, such as COMT, Thymidylatesynthase, PARP, Dihydropteroate synthetase, Farnesyltransferase, GABAtransaminase, Nucleotidyltransferase (Integrase Reverse transcriptase),Protein kinase (Tyrosine-kinase (Janus kinase)); Hydrolases, such asPhosphodiesterase, Acetylcholinesterase, Ribonuclease,Polygalacturonase, Neuraminidase, Alpha-glucosidase, Protease,Exopeptidase (Dipeptidyl peptidase-4, ACE), Endopeptidase (Trypsin,Renin, Matrix metalloproteinase), Histone deacetylase, andBeta-lactamase; and lyases, such as Dopa decarboxylase and Carbonicanhydrase.

Purinoceptor Agonists or Antagonists

Purinergic receptors, also known as purinoceptors, are a family ofplasma membrane molecules that are found in almost all mammaliantissues. Purinergic receptors and signaling have been implicated inlearning and memory, locomotor and feeding behavior, and sleep. Morespecifically, these receptors are involved in several cellularfunctions, including proliferation and migration of neural stem cells,vascular reactivity, apoptosis and cytokine secretion.

P2Y receptors are a family of purinergic G protein-coupled receptors,stimulated by nucleotides such as ATP, ADP, UTP, UDP and UDP-glucose.P2Y receptors are present in almost all human tissues where they exertvarious biological functions based on their G-protein coupling. P2Y2 isa potential drug target for treating cystic fibrosis. Studies have shownthat activating purinoceptors in cystic fibrosis is a possible mechanismto alleviating inflammation and reducing infection. Accordingly,compounds that activate these receptors can be used in combination withthe formulations described herein, which restore and/or maintainhomeostasis in the mucosal membranes of the lungs of cystic fibrosispatients.

P2Y11 is a regulator of immune response, and a common polymorphismcarried by almost 20% of North European Caucasians give increased riskof myocardial infarction, making P2Y11 an interesting drug targetcandidate for treatment of myocardial infarction.

P2Y12 is the target of the anti-platelet drug clopidogrel and otherthienopyridines.

Modulators of these receptors can be used to treat cytotoxic edema,chronic pain, and diabetes. Representative modulators includeclopidogrel, prasugrel and ticlopidine, as well as ticagrelor, all ofwhich are antiplatelet agents that block P2Y12 receptors.

The compounds described herein can be used in combination withpurinoceptor modulators to reduce dosages of these modulators, therebylimiting exposure to toxic drug levels. Additionally, they can be usedin conjunction with other therapies to increase efficacy and/or decreaseside effects.

Cyclooxygenase-2 (COX-2) Inhibitors

COX-2 selective inhibitors are a form of non-steroidal anti-inflammatorydrug (NSAID) that directly target COX-2, an enzyme responsible forinflammation and pain. Targeting selectivity for COX-2 reduces the riskof peptic ulceration, and is the main feature of celecoxib, rofecoxiband other members of this drug class.

By combining the compounds described herein with COX-2 inhibitors, onecan lower the dosage and/or reduce the side effects of these inhibitors.

Bactericidal and Bacteriostatic Agents (“Antibiotics”)

Bactericidals kill bacteria directly, whereas bacteriostatics preventthem from dividing. However, these classifications are based onlaboratory behavior. In practice, both can prevent a bacterialinfection. They work through a variety of mechanisms (e.g., DNA gyraseinhibition, inhibiting bacterial cell wall synthesis, binding to thebacterial ribosomal subunits, and inhibiting protein synthesis). Someexamples of the classes of antibiotics are multiple generations ofcephalosporins, fluoroquinolones, and aminoglycosides.

When the compounds described herein are used in conjunction withantibiotics, they can reduce dosages of the antibiotics, limitingexposure to toxic drug levels and/or the side effects associated withantibiotics (i.e., drug resistance and diarrhea), in some embodiments byhelping wounded mucosal tissues to heal and/or resist further infection.

DNA Gyrase Inhibitors

DNA gyrase, often referred to simply as gyrase, is an enzyme thatrelieves strain while double-stranded DNA is being unwound by helicase.This causes negative supercoiling of the DNA. Bacterial DNA gyrase isthe target of many antibiotics, including nalidixic acid, novobiocin,and ciprofloxacin. Two classes of antibiotics that inhibit gyrase areaminocoumarins (including novobiocin) and the quinolones (includingnalidixic acid and ciprofloxacin).

Natural Killer Cell and Natural Killer T Cell Agonists or Antagonists

Natural killer cells (or NK cells) are a type of cytotoxic lymphocytecritical to the innate immune system. The role NK cells play isanalogous to that of cytotoxic T cells in the vertebrate adaptive immuneresponse. NK cells provide rapid responses to virally infected cells andrespond to tumor formation, acting at around 3 days after infection.Typically immune cells detect MHC presented on infected cell surfaces,triggering cytokine release, causing lysis or apoptosis. NK cells canrecognize stressed cells in the absence of antibodies and MHC, allowingfor a much faster immune reaction. They were named “natural killers”because of the initial notion that they do not require activation inorder to kill cells that are missing “self” markers of majorhistocompatibility complex (MHC) class 1.

Representative NK modulators include cortisol and histone deacetylaseinhibitors (HDACs).

Cathepsin Class Agonists or Antagonists

Cathepsins are proteases which have a vital role in mammalian cellularturnover, including bone resorption. Cathepsins have been implicated incancer, stroke, Alzheimer's disease, arthritis, Ebola, COPD, chronicperiodontitis, pancreatitis, several ocular disorders: keratoconus,retinal detachment, age-related macular degeneration, and glaucoma.

Deficiencies in cathepsin A are linked to multiple forms ofgalactosialidosis. The cathepsin A activity in lysates of metastaticlesions of malignant melanoma is significantly higher than in primaryfocus lysates. Cathepsin A increased in muscles moderately affected bymuscular dystrophy and denervating diseases.

Cathepsin B seems to actually break down the proteins that cause amyloidplaque, the root of Alzheimer's symptoms, and may even be a pivotal partof the natural defense against this disease used by people who do notget it. Overexpression of the encoded protein, which is a member of thepeptidase C1 family, has been associated with esophageal adenocarcinomaand other tumors. Cathepsin B has also been implicated in theprogression of various human tumors including ovarian cancer. CathepsinB is also involved in apoptosis as well as degradation of myofibrillarproteins in myocardial infarction.

Cathepsin D (an aspartyl protease) appears to cleave a variety ofsubstrates such as fibronectin and laminin. Unlike some of the othercathepsins, cathepsin D has some protease activity at neutral pH. Highlevels of this enzyme in tumor cells seems to be associated with greaterinvasiveness.

Cathepsin K is the most potent mammalian collagenase. Cathepsin K isinvolved in osteoporosis, a disease in which a decrease in bone densitycauses an increased risk for fracture. Osteoclasts are the boneresorbing cells of the body, and they secrete cathepsin K in order tobreak down collagen, the major component of the non-mineral proteinmatrix of the bone. Cathepsin K, among other cathepsins, plays a role incancer metastasis through the degradation of the extracellular matrix.The genetic knockout for cathepsin S and K in mice with atherosclerosiswas shown to reduce the size of atherosclerotic lesions. The expressionof cathepsin K in cultured endothelial cells is regulated by shearstress. Cathepsin K has also been shown to play a role in arthritis.

Cathepsin activity has been shown to be modulated or enhanced by theconcentrations of the components of this therapy. This therapy has thepotential to reduce dosages of other therapies, thereby limitingexposure to toxic drug levels. Additionally, it can be used inconjunction with other therapies to increase efficacy and/or decreaseside effects.

Cathepsin inhibitors, particularly cathepsin K inhibitors, have beenused as anti-resorptives for treating osteoporosis, and also for use intreating arthritis, atherosclerosis, high blood pressure, obesity andcancer. One representative inhibitor is Ibandronic acid (INN) oribandronate sodium (USAN), marketed under the trade name Boniva®.

Antioxidant Therapies

An antioxidant is a molecule that inhibits the oxidation of othermolecules. Antioxidants terminate oxidation reactions by removing freeradical intermediates, and inhibit other oxidation reactions. They dothis by being oxidized themselves, so antioxidants are often reducingagents such as thiols, ascorbic acid, or polyphenols. Glutathione,cysteine, and N-acetyl cysteine are representative antioxidants, andadditional antioxidants can be used.

Oxidative stress is thought to contribute to the development of a widerange of diseases, including Alzheimer's disease, Parkinson's disease,the pathologies caused by diabetes, rheumatoid arthritis,neurodegeneration in motor neuron diseases, and certain cancers(including those associated with reactive oxygen species, or “ROS.”

Accordingly, antioxidant therapy can be used to treat, prevent, orreduce the severity of many diseases and conditions (e.g., cancers,cystic fibrosis, autoimmune disorders, organ transplants).

Rho-Associated Kinase Inhibitors

Rho-associated protein kinase (ROCK) is a kinase belonging to the AGC(PKA/PKG/PKC) family of serine-threonine kinases. It is mainly involvedin regulating the shape and movement of cells by acting on thecytoskeleton.

ROCK signaling plays an important role in many diseases, includingdiabetes, neurodegenerative diseases, pulmonary hypertension, andcancer. ROCK inhibitors, such as arachadonic acid, can be used to treatthese diseases, for example, potentially prevent cancer from spreadingby blocking cell migration, stopping cancer cells from spreading intoneighboring tissue.

Myosin Inhibitors

Myosins comprise a family of ATP-dependent motor proteins and are bestknown for their role in muscle contraction and their involvement in awide range of other eukaryotic motility processes. Myosins function in awide variety of cellular processes, from intracellular trafficking tocell motility, and are implicated in several human diseases (e.g.,cancer, hypertrophic cardiomyopathy, deafness and many neurologicaldisorders). Myosin inhibitors can therefore be used to treat these typesof diseases. Representative myosin inhibitors include N-benzyl-p-toluenesulphonamide (BTS); 2,3-Butanedione monoxime (BDM); Pentachloropseudilin(PCIP); Pentabromopseudilin (PBP); MyoVin-1; and 2,4,6-Triiodophenol.

Phosphatidylinositol 3 Kinase Inhibitors and Related Molecules

Phosphatidylinositide 3-kinases (PI 3-kinases, PI3Ks, PI(3)Ks, orPI-3Ks) are a family of enzymes involved in cellular functions such ascell growth, proliferation, differentiation, motility, survival andintracellular trafficking, which in turn are involved in cancer.

Wortmannin and LY294002 are broad inhibitors against PI 3-kinases. Otherrepresentative PI 3 Kinase inhibitors include the class I PI 3-kinase,p110δ isoform specific inhibitors, IC486068 and IC87114 (ICOSCorporation), as well as GDC-0941 (Genentech), a highly selectiveinhibitor of p110α with little activity against mTOR.

Nitric Oxide Synthase Agonists or Antagonists, Nitric Oxide Agonists orAntagonists

Nitric oxide synthases (NOSs) are a family of enzymes catalyzing theproduction of nitric oxide (NO) from L-arginine. NO is an importantcellular signaling molecule. It helps modulate vascular tone, insulinsecretion, airway tone, and peristalsis, and is involved in angiogenesisand neural development. Inhibitors of these enzymes can help treatstroke.

Representative NOS inhibitors include guanidino aminoacids,aminoguanidine, NG-iminoethyl-L-lysine (L-NIL), NG-iminoethyl-Lornithine(L-NIO), the bis-isothioureas (PBITU), 1400W(N-[3-(aminomethyl)benzyl]acetamidine), GW273629 and GW274150,7-nitroindinazole (7-NI), tri(fluoromethylphenyl)imidazole (TRIM), ARL17477, AR-R18512, BN 80933, S-ethyl and S-methyl thiocitrulline andvinyl L-NIO.

Ion Channel Function or Trafficking Modulators

Ion channels are pore-forming membrane proteins whose functions includeestablishing a resting membrane potential, shaping action potentials andother electrical signals by gating the flow of ions across the cellmembrane, controlling the flow of ions across secretory and epithelialcells, and regulating cell volume.

Ion channels are present in the membranes of all cells. Ion channels areconsidered to be one of the two traditional classes of ionophoricproteins, with the other class known as ion transporters (including thesodium-potassium pump, sodium-calcium exchanger, and sodium-glucosetransport proteins, amongst others).

Channels differ with respect to the ion they let pass (for example, Na+,K+, Cl—), the ways in which they may be regulated, the number ofsubunits of which they are composed and other aspects of structure.

Representative channel blockers include:

Tetrodotoxin (TTX), used by puffer fish and some types of newts fordefense. It blocks sodium channels.

Saxitoxin, is produced by a dinoflagellate also known as “red tide.” Itblocks voltage dependent sodium channels.

Conotoxin, is used by cone snails to hunt prey.

Lidocaine and Novocaine belong to a class of local anesthetics whichblock sodium ion channels.

Dendrotoxin is produced by mamba snakes, and blocks potassium channels.

Iberiotoxin is produced by Buthus tamulus (Eastern Indian scorpion) andblocks potassium channels.

Heteropodatoxin is produced by Heteropoda venatoria (brown huntsmanspider or laya) and blocks potassium channels.

CFTR is an ABC transporter-class ion channel that transports chlorideand thiocyanate ions across epithelial cell membranes. Mutations of theCFTR gene affect functioning of the chloride ion channels in these cellmembranes, leading to cystic fibrosis and congenital absence of the vasdeferens. This therapy has the potential to reduce dosages of othertherapies that facilitate the expression, transport, or function ofCFTR, thereby limiting exposure to toxic drug levels. Additionally, itcan be used in conjunction with these and other therapies to increaseefficacy and/or decrease side effects.

Surfactant Therapies, in Particular, Lung (Pulmonary) Surfactants

Surfactants are compounds that lower the surface tension or interfacialtension between two liquids or between a liquid and a solid. Surfactantsmay act as detergents, wetting agents, emulsifiers, foaming agents, ordispersants.

Pulmonary surfactant are surface-active lipoprotein complexes(phospholipoprotein) formed by type II alveolar cells. The proteins andlipids that comprise the surfactant have both a hydrophilic region and ahydrophobic region. By adsorbing to the air-water interface of alveoliwith the hydrophilic head groups in the water and the hydrophobic tailsfacing towards the air, the main lipid component of surfactant,dipalmitoylphosphatidylcholine (DPPC), reduces surface tension.

In the lungs, surfactant reduces the surface tension and helps tomaximize the surface area available for gas exchange. Without adequatesurfactant, a baby works much harder to breathe, becomes exhausted, anddoes not get enough oxygen. There are various pulmonary disordersassociated with not having sufficient amounts of pulmonary surfactants,including infant respiratory distress syndrome (IRDS), hyaline membranedisease, congenital surfactant deficiency, and pulmonary alveolarproteinosis. Synthetic lung surfactants can be administered to overcomethe deficiency. Perhaps the most widely-administered pulmonarysurfactant is Curosurf® (Cornerstone Therapeutics).

When a lung surfactant is combined with the therapeutic agents describedherein, the resulting therapy can reduce dosages of other therapies,thereby limiting exposure to toxic drug levels. Additionally, it can beused in conjunction with other therapies to increase efficacy and/ordecrease side effects.

Cannabinoid Receptor Modulators

The cannabinoid receptors are a class of cell membrane receptors underthe G protein-coupled receptor superfamily. Cannabinoid receptors areactivated by three major groups of ligands, endocannabinoids (producedby the mammalian body), plant cannabinoids (such as THC, produced by thecannabis plant) and synthetic cannabinoids (such as HU-210).

Synthetic THC is prescribed today, under the INN dronabinol or the brandname Marinol, to treat vomiting and for enhancement of appetite, mainlyin AIDS patients. Several synthetic cannabinoids have been shown to bindto the CB2 receptor with a higher affinity than to the CB1 receptor.Most of these compounds exhibit only modest selectivity. One of thedescribed compounds, a classical THC-type cannabinoid, L-759,656, inwhich the phenolic group is blocked as a methyl ether, has a CB1/CB2binding ratio >1000.

Certain tumors, especially gliomas, express CB2 receptors.Δ9-tetrahydrocannabinol and WIN-55, 212-2, two non-selective cannabinoidagonists, induce the regression or eradication of malignant brain tumorsin rats and mice. CB2 selective agonists are effective in the treatmentof pain, various inflammatory diseases in different animal models,osteoporosis and atherosclerosis. CB1 selective antagonists havepreviously been used for weight reduction and smoking cessation.Activation of CB1 provides neuroprotection after brain injury.

Representative cannabinoid receptor modulators include Anandamide,N-Arachidonoyl dopamine, 2-Arachidonoylglycerol, 2-Arachidonyl glycerylether, Tetrahydrocannabinol, Epigallocatechin gallate (EGCG) 33, AM-1221(1-[(N-methylpiperidin-2-yl)methyl]-2-methyl-3-(naphthalen-1-oyl)-6-nitroindole),AM-1235(-[(5-fluoropentyl)-6-nitro-1H-indol-3-yl]-(naphthalen-1-yl)methanone),AM-2232 (5-(3-(1-naphthoyl)-1H-indol-1-yl)pentanenitrile), UR-144((1-pentylindol-3-yl)-(2,2,3,3-tetramethylcyclopropyl)methanone),JWH-007 (1-pentyl-2-methyl-3-(1-naphthoyl)indole), JWH-015((2-Methyl-1-propyl-1H-indol-3-yl)-1-naphthalenylmethanone) and JWH-018(1-pentyl-3-(1-naphthoyl)indole) (Clemson University).

Complement System Inhibitors

The complement system is a biochemical cascade that attacks the surfacesof foreign cells. It contains over 20 different proteins and is namedfor its ability to “complement” the killing of pathogens by antibodies.Complement is the major humoral component of the innate immune response.

The complement system helps or “complements” the ability of antibodiesand phagocytic cells to clear pathogens from an organism. It is part ofthe immune system (called the innate immune system) that is notadaptable and does not change over the course of an individual'slifetime. However, it can be recruited and brought into action by theadaptive immune system. It is thought that the complement system mightplay a role in many diseases with an immune component, such asBarraquer-Simons Syndrome, asthma, lupus erythematosus,glomerulonephritis, various forms of arthritis, autoimmune heartdisease, multiple sclerosis, inflammatory bowel disease, paroxysmalnocturnal hemoglobinuria, atypical hemolytic uremic syndrome andischemia-reperfusion injuries and rejection of transplanted organs. Thecomplement system is also becoming increasingly implicated in diseasesof the central nervous system such as Alzheimer's disease and otherneurodegenerative conditions such as spinal cord injuries.

Deficiencies of the terminal pathway predispose to both autoimmunedisease and infections (particularly Neisseria meningitidis, due to therole that the membrane attack complex plays in attacking Gram-negativebacteria). Recent research has suggested that the complement system ismanipulated during HIV/AIDS to further damage the body.

Representative complement inhibitors include Soliris® (eculizumab,Alexion) which works by inhibiting the complement protein C5, which inturn acts at a relatively late stage in the complement cascade.

When complement inhibitors are combined with the therapeutic agentsdescribed herein, the combined therapy can reduce dosages of othertherapies, thereby limiting exposure to toxic drug levels. Additionally,it can be used in conjunction with other therapies to increase efficacyand/or decrease side effects.

G Protein-Coupled Receptor Agonists or Antagonists

G protein coupled receptors (GPCRs), also known as seven-transmembranedomain receptors, 7TM receptors, heptahelical receptors, serpentinereceptor, and G protein-linked receptors (GPLR), constitute a largeprotein family of receptors that sense molecules outside the cell andactivate inside signal transduction pathways and, ultimately, cellularresponses. An example of a GPCR is the cannabinoid receptors CB1 andCB2.

The ligands that bind and activate these receptors includelight-sensitive compounds, odors, pheromones, hormones, andneurotransmitters, and vary in size from small molecules to peptides tolarge proteins. G protein-coupled receptors are involved in manydiseases, and are also the target of approximately 40% of all modernmedicinal drugs. There are two principal signal transduction pathwaysinvolving the G protein-coupled receptors: the cAMP signal pathway andthe phosphatidylinositol signal pathway. When a ligand binds to the GPCRit causes a conformational change in the GPCR, which allows it to act asa guanine nucleotide exchange factor (GEF). The GPCR can then activatean associated G-protein by exchanging its bound GDP for a GTP. TheG-protein's α subunit, together with the bound GTP, can then dissociatefrom the β and γ subunits to further affect intracellular signalingproteins or target functional proteins directly depending on the αsubunit type.

This therapy has the potential to reduce dosages of other therapies,thereby limiting exposure to toxic drug levels. Additionally, it can beused in conjunction with other therapies that modulate any or allcomponents of GPCRs to increase efficacy and/or decrease side effects.

IgE Receptor Antagonists, G Protein-Coupled Receptor Agonists orAntagonists

Immunoglobulin E (IgE) is a class of antibody (or immunoglobulin (Ig)“isotype”). IgE exists as monomers consisting of two heavy chains (εchain) and two light chains, with the ε chain containing 4 Ig-likeconstant domains (Cε1-Cε4). IgE's main function is immunity to parasitessuch as parasitic worms. IgE also plays an essential role in type Ihypersensitivity, which manifests various allergic diseases, such asallergic asthma, allergic rhinitis, food allergy, and some types ofchronic urticaria and atopic dermatitis. IgE also plays a pivotal rolein allergic conditions, such as anaphylactic reactions to certain drugs,bee stings, and antigen preparations used in specific desensitizationimmunotherapy.

Currently, allergic diseases and asthma are usually treated with one ormore of the following drugs: (1) antihistamines and antileukotrienes,which antagonize the inflammatory mediators histamine and leukotrienes,(2) local or systemic (oral or injectable) corticosteroids, whichsuppress a broad spectrum of inflammatory mechanisms, and (3) short orlong-acting bronchodilators, which relax smooth muscle of constrictedairway in asthma.

Chemokines, Chemokine Receptor Agonists or Antagonists

Chemokines are a family of small cytokines, or signaling proteinssecreted by cells. Their name is derived from their ability to inducedirected chemotaxis in nearby responsive cells; they are chemotacticcytokines. Some chemokines are considered pro-inflammatory and can beinduced during an immune response to recruit cells of the immune systemto a site of infection, while others are considered homeostatic and areinvolved in controlling the migration of cells during normal processesof tissue maintenance or development.

Chemokines have been classified into four main subfamilies: CXC, CC,CX3C and XC. All of these proteins exert their biological effects byinteracting with G protein-linked transmembrane receptors calledchemokine receptors, which are selectively found on the surfaces oftheir target cells. It has been suggested that blockage of chemokinefunction using chemokine inhibitors should not have a detrimentaltoxicological effect.

The cyclic peptide NR58-3.14.3 is a powerful anti-inflammatory agent,inhibiting inflammation in a number of disease models such asatherosclerosis, ischemia, lung disease, surgical adhesions,endometriosis, and pulmonary graft-versus-host disease. Cyclic peptideNR58-3.14.3 has also been shown to inhibit HIV replication.

Cytokines, and Cytokine Receptor Agonists or Antagonists

Cytokines such as tumor necrosis factor-a (TNF-a) and interleukin 1(IL-1) are important mediators of inflammation and tissue damage inanimal models of inflammatory arthritis and in patients with activerheumatoid arthritis (RA).

Inhibitors of these cytokines can be used to treat RA. Several widelyused drugs, including corticosteroids, methotrexate, and cyclosporin A,are known to block cytokine production from macrophages and T cells.Representative inhibitors include Etanercept, is a recombinant fusionprotein of the soluble type II TNF receptor on a human IgG1 backbone,infliximab, a chimeric anti-TNF-a monoclonal antibody containing amurine TNF-a binding region and human IgG1 backbone. Anakinra is arecombinant human IL-1 receptor antagonist (IL-1Ra) that binds avidly totype 1 IL-1 receptors but does not stimulate any intracellularresponses.

Arachidonic Acid Agonists or Antagonists

Arachidonic acid (AA, sometimes ARA) is a polyunsaturated omega-6 fattyacid 20:4(ω-6). Flavonoids have been shown to have anti-inflammatoryactivity and to inhibitor arachidonic acid metabolism.

Inflammatory Mediators

Inflammation is part of the complex biological response of vasculartissues to harmful stimuli, such as pathogens, damaged cells orirritants. Inflammation is a protective attempt by the organism toremove the injurious stimuli and to initiate the healing process.However, inflammatory responses are often accompanied by unwanted sideeffects (e.g., histamine release during an allergic response causingsneezing).

Inflammatory mediators are soluble, diffusible molecules that actlocally at the site of tissue damage and infection, and at more distantsites. They can be divided into exogenous and endogenous mediators.

Endogenous inflammatory mediators produced from within the (innate andadaptive) immune system itself, as well as other systems. Examples ofendogenous inflammatory mediators are nitric oxide synthase agonists orantagonists, nitric oxide agonists or antagonists, arachidonic acidagonists or antagonists, histamine or leukotriene agonists orantagonists.

Bacterial products and toxins can act as exogenous inflammatorymediators, an example of which is endotoxin, or LPS of Gram-negativebacteria. Endotoxin can trigger complement activation, resulting in theformation of anaphylatoxins C3a and C5a which cause vasodilation andincrease vascular permeability. Endotoxin also activates the Hagemanfactor, leading to activation of both the coagulation and fibrinolyticpathways as well as the kinin system. In addition, endotoxin elicit Tcell proliferation, and have been described as superantigen for T cells.

Mononuclear phagocytes (monocytes and macrophages) are central toinflammation, as they produce many components which participate in orregulate the different plasma enzyme systems, and hence the mediators ofthe inflammatory response. They are also actively phagocytic and areinvolved in microbial killing, as are neutrophils. While the latter canbe thought of as short-lived kamikaze cells that need to be continuallyreplaced from the bone marrow, mononuclear phagocytes are long-lived andsome can proliferate in situ. Other cells such as mast cells andbasophils are much less phagocytic, but together with platelets, thesecells are particularly important for secretion of vasoactive mediators.The function of these cell types is at least partially under the controlof cytokines. All inflammatory cells have receptors for Fc domains ofimmunoglobulins and for complement components, and they possessspecialized granules containing an immerse variety of products that arereleased perhaps by common mechanisms. Cytotoxic T lymphocytes and NKcells, in general, also possess granules which are important for theircytotoxic function. In general, lymphocytes are involved in the adaptiveresponse to inflammation, and the early events of inflammation aremediated in part by molecules produced by cells of the innate arm of theimmune system.

Early phase mediators are produced by mast cells and platelets. They areespecially important in acute inflammation and include mainly histamine,serotonin and other vasoactive substances. Platelets may contribute toinflammatory responses resulting as a consequence of tissue injury,through a variety of mechanisms including the release of vasoactiveamines and other permeability factors, the release of lysosomal enzymes,the release of coagulation factors which lead to localized andgeneralized fibrin deposition, and the formation of platelet aggregatesor trombi which result in the blocking of vessels and capillaries. Tothe early phase mediators also belong chemoattractants (e.g. C5a) andcytokines such as IL-1, IL-6, and TNF-α.

Late phase mediators are responsible for the regulation of vascularevents later—from about 6-12 hours after initiation of inflammation. Thelater vascular events are mediated, at least in part, by products ofarachidonic acid.

When inflammatory mediators are combined with the therapeutic agentsdescribed herein, the combined therapy can reduce dosages of othertherapies, thereby limiting exposure to toxic drug levels. Additionally,it can be used in conjunction with other therapies that modulate any orall components of GPCRs to increase efficacy and/or decrease sideeffects.

STAT6 Inhibitors

Interleukin-4 (IL-4) and IL-13 have a central role in the development ofasthma through activation of the signal transducer and activator oftranscription-6 (STAT6). STAT6 inhibitors can be used to treat asthma.Representative inhibitors include Vorinostat and other HDAC inhibitors.

Histamine or Leukotriene Agonists or Antagonists

Histamine is an organic nitrogen compound involved in local immuneresponses as well as regulating physiological function in the gut andacting as a neurotransmitter. Histamine triggers the inflammatoryresponse.

Histamine affects mucous membranes in three main ways, includingsneezing due to histamine-associated sensory neural stimulation,hyper-secretion from glandular tissue, and nasal congestion due tovascular engorgement associated with vasodilation and increasedcapillary permeability.

Histamine antagonists (or blockers) can be used to inhibit theinflammatory response associated with histamine, for treating allergysymptoms and preventing anaphylaxis.

H1-receptor antagonists (anti-histamines) are actually inverse agonistsat the histamine H1-receptor. Clinically, H1 antagonists are used totreat allergic reactions. Examples include Acrivastine, Azelastine,Bilastine, Brompheniramine, Buclizine, bromodiphenhydramine,Carbinoxamine, Cetirizine (Metabolite of Hydroxyzine), Chlorpromazine(antipsychotic), Cyclizine, Chlorpheniramine, Chlorodiphenhydramine,Clemastine, Cyproheptadine, Desloratadine, Dexbrompheniramine,Dexchlorpheniramine, Dimenhydrinate (most commonly used as anantiemetic), Dimetindene, Diphenhydramine (Benadryl), Doxylamine (mostcommonly used as an OTC sedative), Ebastine, Embramine, Fexofenadine(Allegra), Hydroxyzine (Vistaril), Levocetirizine, Loratadine(Claritin), Meclozine (most commonly used as an antiemetic), Mirtazapine(primarily used to treat depression, also has antiemetic andappetite-stimulating effects), Olopatadine (used locally), Orphenadrine(a close relative of diphenhydramine used mainly as a skeletal musclerelaxant and anti-Parkinsons agent), Phenindamine, Pheniramine,Phenyltoloxamine, Promethazine, Pyrilamine, Quetiapine (antipsychotic;trade name Seroquel), Rupatadine, Tripelennamine, and Triprolidine.

H2-receptor antagonists are also inverse agonists and not trueantagonists. They act on H2 histamine receptors found principally in theparietal cells of the gastric mucosa, which are part of the endogenoussignaling pathway for gastric acid secretion. Normally, histamine actson H2 to stimulate acid secretion; drugs that block 1-12 signaling thusreduce the secretion of gastric acid. H2 antagonists are amongfirst-line therapy to treat gastrointestinal conditions including pepticulcers and gastroesophageal reflux disease. Representative H2-receptorantagonists include Cimetidine, Famotidine, Lafutidine, Nizatidine,Ranitidine, and Roxatidine.

Calcineurin Agonists or Antagonists

Calcineurin (CN) is a protein phosphatase also known as proteinphosphatase 3, and calcium-dependent serine-threonine phosphatase. Itactivates the T cells of the immune system, and can be blocked by drugscalled calcineurin inhibitors. Representative compounds includecyclosporine, pimecrolimus and tacrolimus.

Calcineurin activates nuclear factor of activated T cell, cytoplasmic(NFATc), a transcription factor, by dephosphorylating it. The activatedNFATc is then trans located into the nucleus, where it upregulates theexpression of interleukin 2 (IL-2), which, in turn, stimulates thegrowth and differentiation of T cell response.

When an antigen-presenting cell interacts with a T cell receptor on Tcells, there is an increase in the cytoplasmic level of calcium,which[3] activates calcineurin, by binding a regulatory subunit andactivating calmodulin binding. Calcineurin induces differenttranscription factors (NFATs) that are important in the transcription ofIL-2 genes. IL-2 activates T-helper lymphocytes and induces theproduction of other cytokines. In this way, it governs the action ofcytotoxic lymphocytes. The amount of IL-2 being produced by the T-helpercells is believed to influence the extent of the immune responsesignificantly.

Calcineurin antagonists can be used to treat rheumatic diseases, such asRA, alone or in combination with methotrexate, psoriatic arthritis,psoriasis, acute ocular Behcet's disease, juvenile idiopathic arthritis,adult and juvenile polymyositis and dermatomyositis, adult and juvenilesystemic lupus erythematosus, adult lupus membranous nephritis, systemicsclerosis, aplastic anemia, steroid-resistant nephrotic syndrome, atopicdermatitis, severe ulcerative colitis, pemphigus vulgaris, myastheniagravis, and dry eye disease, with or without Sjögren's syndrome.

Calcineurin is also linked to receptors for several brain chemicalsincluding NMDA, dopamine and GABA, and inhibitors can be used to treatschizophrenia, impairment in working memory, attention deficits,aberrant social behavior, and several other abnormalities characteristicof schizophrenia.

Calcineurin along with NFAT, can improve the function of diabetics'pancreatic beta cells. Calcineurin/Nfat signaling is required forperinatal lung maturation and function.

Anti-HIV Agents

In some embodiments, the method comprises co-administration of anantiretroviral agent, and particularly an agent used for the treatmentof HIV infection such as Zidovudine (AZT), Abacavir, Emtricitabine(FTC), Lamivudine (3TC), Didanosine (ddI), Stavudine (d4T), Zalcitabine(ddC), Nevirapine, Efavirenz, Delavirdine, Tenofovir, Enfuvirtide (T20),Maraviroc (CCR5), Lopinavir, Atazanavir, Fosamprenvir, Amprenavir,Saquinavir, Indinavir, Nelfinavir, Raltegravir, and Elvitegravir.

One or more, preferably one to four, antiviral agents useful inanti-HIV-1 therapy may be used. The antiviral agents contemplated foruse comprise nucleoside and nucleotide reverse transcriptase inhibitors,non-nucleoside reverse transcriptase inhibitors, protease inhibitors andother antiviral drugs listed below not falling within theseclassifications. In particular, the combinations known as HAART arecontemplated for use.

The term “nucleoside and nucleotide reverse transcriptase inhibitors”(“NRTI”s) as used herein means nucleosides and nucleotides and analoguesthereof that inhibit the activity of HIV-1 reverse transcriptase, theenzyme which catalyzes the conversion of viral genomic HIV-1 RNA intoproviral HIV-1 DNA.

Typical suitable NRTIs include zidovudine (AZT) available under theRETROVIR tradename from Glaxo-Wellcome Inc., Research Triangle, N.C.27709; didanosine (ddI) available under the VIDEX tradename fromBristol-Myers Squibb Co., Princeton, N.J. 08543; zalcitabine (ddC)available under the HMD tradename from Roche Pharmaceuticals, Nutley,N.J. 07110; stavudine (d4T) available under the ZERIT trademark fromBristol-Myers Squibb Co., Princeton, N.J. 08543; lamivudine (3TC)available under the EPIVIR tradename from Glaxo-Smith Kline Triangle,N.C. 27709; abacavir (1592U89) disclosed in WO96/30025 and availableunder the ZIAGEN trademark from Glaxo-Wellcome Research Triangle, N.C.27709; adefovir dipivoxil [bis(POM)-PMEA] available under the PREVONtradename from Gilead Sciences, Foster City, Calif. 94404; lobucavir(BMS-180194), a nucleoside reverse transcriptase inhibitor disclosed inEP-0358154 and EP-0736533 and under development by Bristol-Myers Squibb,Princeton, N.J. 08543; BCH-10652, a reverse transcriptase inhibitor (inthe form of a racemic mixture of BCH-10618 and BCH-10619) underdevelopment by Biochem Pharma, Laval, Quebec H7V, 4A7, Canada;emitricitabine [(−)-FTC] available from Gilead under the trade nameEmtrivia™; beta-L-FD4 (also called beta-L-D4C and namedbeta-L-2′,3′-dicleoxy-5-fluoro-cytidene) (Vion Pharmaceuticals, NewHaven Conn. 0651 1); DAPD, the purine nucleoside,(−)-beta-D-2,6,-diamino-purine dioxolane disclosed in EP 0656778(Triangle Pharmaceuticals, Durham, N.C.); and lodenosine (FddA),9-(2,3-dideoxy-2-fluoro-b-D-threo-pentofuranosyl)adenine, an acid stablepurine-based reverse transcriptase inhibitor under development by U.S.Bioscience Inc., West Conshohoken, Pa. 19428. The term “non-nucleosidereverse transcriptase inhibitors” (“NNRTI11S) as used herein meansnon-nucleosides that inhibit the activity of HIV-1 reversetranscriptase.

Typical suitable NNRTIs include nevirapine (BI-RG-587) available underthe VIRAMUNE tradename from Boehringer Ingelheim, the manufacturer forRoxane Laboratories, Columbus, Ohio 43216; delaviradine (BHAP, U-90152)available under the RESCRIPTOR tradename from Pharmacia & Upjohn Co.,Bridgewater N.J. 08807; efavirenz (DMP-266) a benzoxazin-2-one disclosedin WO94/03440 and available under the SUSTIVA tradename from BristolMyers Squibb in the US and Merck in Europe; PNU-142721, afuropyridine-thio-pyrimide under development by Pharmacia and Upjohn,Bridgewater N.J. 08807; AG-1549 (formerly Shionogi # S-1153);5-(3,5-dichlorophenyl)-thio-4-isopropyl-1-(4-pyridyl)methyl-1H-imidazol-2-ylmethylcarbonate disclosed in WO 96/10019 and under clinical development byAgouron Pharmaceuticals, Inc., LaJolla Calif. 92037-1020; MKC-442(1-(ethoxy-methyl)-5-(1-methylethyl)-6-(phenylmethyl)-(2,4(1H,3H)-pyrimidinedione)discovered by Mitsubishi Chemical Co. and under development by TrianglePharmaceuticals, Durham, N.C. 27707; (+)-calanolide A (NSC-675451) andB, coumarin derivatives disclosed in NIH U.S. Pat. No. 5,489,697,licensed to Med Chem Research, which is co-developing (+) calanolide Awith Vita-Invest as an orally administrable product; and etravirine(TMC-125, Intelence) marketed by Tibotec.

The term “protease inhibitor” (“PI”) as used herein means inhibitors ofthe HIV-1 protease, an enzyme required for the proteolytic cleavage ofviral polyprotein precursors (e.g., viral GAG and GAG Pol polyproteins),into the individual functional proteins found in infectious HIV-1. HIVprotease inhibitors include compounds having a peptidomimetic structure,high molecular weight (7600 daltons) and substantial peptide character,e.g. CRIXIVAN (available from Merck) as well as nonpeptide proteaseinhibitors e.g., VIRACEPT (available from Agouron).

Typical suitable PIs include saquinavir (Ro 31-8959) available in hardgel capsules under the INVIRASE tradename and as soft gel capsules underthe FORTOVASE tradename from Roche Pharmaceuticals, Nutley, N.J.07110-1199; ritonavir (ABT-538) available under the NORVIR tradenamefrom Abbott Laboratories, Abbott Park, Ill. 60064; indinavir (MK-639)available under the CRIXIVAN tradename from Merck & Co., Inc., WestPoint, Pa. 19486-0004; nelfnavir (AG-1343) available under the VIRACEPTtradename from Agouron Pharmaceuticals, Inc., LaJolla Calif. 92037-1020;amprenavir (141W94), tradename AGENERASE, a non-peptide proteaseinhibitor under development by Vertex Pharmaceuticals, Inc., Cambridge,Mass. 02139-4211 and available from Glaxo-Wellcome, Research Triangle,N.C. under an expanded access program; lasinavir (BMS-234475) availablefrom Bristol-Myers Squibb, Princeton, N.J. 08543 (originally discoveredby Novartis, Basel, Switzerland (CGP-61755); DMP-450, a cyclic ureadiscovered by Dupont and under development by Triangle Pharmaceuticals;BMS-2322623, an azapeptide under development by Bristol-Myers Squibb,Princeton, N.J. 08543, as a 2nd-generation HIV-1 PI; ABT-378 underdevelopment by Abbott, Abbott Park, Ill. 60064; AG-1549 an orally activeimidazole carbamate discovered by Shionogi (Shionogi #S-1153) and underdevelopment by Agouron Pharmaceuticals, Inc., LaJolla Calif. 92037-1020;atazanavir; tipranavir; and darunavir.

Other antiviral agents include CXCR4 antagonists, enfuvirtide,hydroxyurea, ribavirin, IL-2, IL-12, pentafuside and Yissum Project No.11607. Hydroxyurea (Droxia), a ribonucleoside triphosphate reductaseinhibitor, the enzyme involved in the activation of T-cells, wasdiscovered at the NCI and is under development by Bristol-Myers Squibb;in preclinical studies, it was shown to have a synergistic effect on theactivity of didanosine and has been studied with stavudine. IL-2 isdisclosed in Ajinomoto EP-0142268, Takeda EP-0176299, and Chiron U.S.Pat. Nos. RE 33,653, 4,530,787, 4,569,790, 4,604,377, 4,748,234,4,752,585, and 4,949,314, and is available under the PROLEUKIN(aldesleukin) tradename from Chiron Corp., Emeryville, Calif. 94608-2997as a lyophilized powder for IV infusion or sc administration uponreconstitution and dilution with water; a dose of about 1 to about 20million ILJ/day, sc is preferred; a dose of about 15 million IU/day, scis more preferred. IL-12 is disclosed in WO96/25171 and is availablefrom Roche Pharmaceuticals, Nutley, N.J. 07110-1199 and American HomeProducts, Madison, N.J. 07940; a dose of about 0.5 microgram/kg/day toabout 10 microgram/kg/day, sc is preferred. Enfuvirtide (DP-178, T-20) a36-amino acid synthetic peptide, is disclosed in U.S. Pat. No.5,464,933; enfuvirtide acts by inhibiting fusion of HIV-1 to targetmembranes. Enfuvirtide (3-100 mg/day) is given as a continuous scinfusion or injection together with efavirenz and 2 Pi's to HIV-1positive subjects refractory to a triple combination therapy; use of 100mg/day is preferred. Yissum Project No. 11607, a synthetic protein basedon the HIV-1 Vif protein, is under development by Yissum ResearchDevelopment Co., Jerusalem 91042, Israel. Ribavirin,1-.beta.-D-ribofuranosyl-1H-1,2,4-triazole-3-carboxamide, is availablefrom ICN Pharmaceuticals, Inc., Costa Mesa, Calif.; its manufacture andformulation are described in U.S. Pat. No. 4,211,771; the integraseinhibitor raltegravir available from Merck under the tradenameIsentress™; elvitegravir an intergrase inhibitor under development byGilead Sciences; the HIV-1Gag maturation inhibitor berivimat underdevelopment (Phase lib) by Panacos Pharmaceuticals.

The term “anti-HIV-1 therapy” as used herein means any anti-HIV-1 drugfound useful for treating HIV-1 infections in man alone, or as part ofmultidrug combination therapies, especially the HAART triple andquadruple combination therapies. Typical suitable known anti-HIV-1therapies include, but are not limited to multidrug combinationtherapies such as (i) at least three anti-HIV-1 drugs selected from twoNRTIs, one PI, a second PI, and one NNRTI; and (ii) at least twoanti-HIV-1 drugs selected from NNRTIs and PIs. Typical suitableHAART—multidrug combination therapies include: (a) triple combinationtherapies such as two NRTIs and one PI; or (b) two NRTIs and one NNRTI;and (c) quadruple combination therapies such as two NRTIs, one PI and asecond PI or one NNRTI. In treatment of naive patients, it is preferredto start anti-HIV-1 treatment with the triple combination therapy; theuse of two NRTIs and one NNRTI or two NRTIs and one PI is preferred ifthere is intolerance to NNRTI. Drug compliance is essential. The CD4+and HIV-1-RNA plasma levels should be monitored every 3-6 months. Shouldviral load plateau, a fourth drug, e.g., one PI, one NNRTI or integraseinhibitor could be added.

Particularly where the compositions include these additional therapeuticagents, the compositions can be used to treat mucosal diseases,pulmonary diseases, autoimmune diseases (including multiple sclerosis,Crohn's disease, ulcerative colitis, lupus, inflammatory bowel syndrome,irritable bowel syndrome, etc.), infectious diseases (e.g., HIV),Alzheimer's disease, and the like.

The compositions and formulations of this invention can be administeredto an immunocompromised subject, a transplant recipient, a subjectundergoing chemotherapy, a subject at increased risk of an opportunisticinfection, a subject infected by HIV, a subject that has acquiredimmunodeficiency syndrome (AIDS), etc., in order to treat and/or preventdiseases and disorders associated with such an immunocompromised orvulnerable status (e.g., Pneumocystis jirovecii pneumonia (PCP),cytomegalovirus (CMV) infection, Mycobacterium avium-intracellulare (MAIor MAC) infection, infection by Mycobacterium other than tuberculosis(MOTT), etc.). The compositions and formulations of this invention canbe administered in an amount that restores and/or maintains ahomeostatic environment in the mucosal membranes of such a subject totreat, prevent or reduce the severity of PCP, CMV infection, MAIinfection, MOTT infection and the like.

In some aspects of these embodiments, a single formulation includes thetherapeutic agent(s) and the glutathione, organic acid and buffer, andin other aspects of these embodiments, the therapeutic agents arepresent in a first formulation and the glutathione, organic acid andbuffer are present in a second formulation. As such, the compositionscan be used in combination or ‘kit’ therapies. The formulation with thetherapeutic agent can be present in oral, injectable, or inhaled forms,and the glutathione, organic acid and buffer present in an inhaled(e.g., pulmonary or intranasal) formulation.

Where the therapeutic agent is present, the resulting new formulationscan provide new applications for the therapeutic agent, enhance theefficacy of the therapeutic agent, reduce unwanted side effectsassociated with the therapeutic agent, and/or reduce the dose of thetherapeutic agent.

While not wishing to be bound by a particular theory, it is believedthat the formulations disclosed herein are effective in achieving andmaintaining either a normal lung mucosa, or at least a more normal lungmucosa, which is an important factor in maintaining lung health. Drugsadministered to the lungs are often associated with certain sideeffects, in some cases because of dosage, and in other cases becausethey damage the lung tissue. In some embodiments, therapeutic agentscombined with the formulations disclosed herein are effective at lowerdoses, and at such lower doses, the incidence of side effects can bereduced. In other embodiments, where the therapeutic agent interactsunfavorably with lung tissue, the formulations described herein can helpto restore homeostasis to the lung tissue, and thus help minimize oreliminate damage caused by the therapeutic agents.

Representative therapeutic agents that can be combined with thecompositions and formulations of this invention include, but are notlimited to, Fluticasone (for example, sold as Flovent diskus 50 or asFlonase, GlaxoSmithKline), Budesonide (for example, sold as Pulmicortrespules or Rhinocort by Astra Zeneca (“AZ”), Mometasone (sold asNasonex as a spray, or as Asmanex Twisthaler by Merck/S-P), Ciclesonide(sold as Alvesco or Omnaris by Takeda Pharmaceuticals), Flunisolide(sold as Aerobid by Roche Palo or by Aerospan HFA by GSK),Beclomethasone (sold as Qvar or Onasl by Teva Pharmaceuticals),Albuterol (sold as ProAir HFA by Teva and as Ventolin HFA by GSK),Levalbuterol (sold as Xopenex by Sunovion), Ipratropium (sold asAtrovent by BI), Tiotropium (sold as Spiriva by BI), Salmeterol (sold asSerevent by GSK), Formoterol (sold as Foradil by Novartis and asPerforomist by Dey Pharma), Arformoterol (sold as Brovana by Sunovion),Indacaterol (sold as Arcapta by Novartis), Aclidinium (sold as Tudorzaby Forest Labs), Pirbuterol (sold as Maxair by Medicis).

To treat pulmonary infections, the formulations described herein can becombined with antibiotics, whether in the same inhaled formulation(i.e., via pulmonary delivery or intranasal delivery), or where theantibiotics are administered by another route, such as the oral orinjectable routes. Representative antibiotics useful in treatingpulmonary infections include, but are not limited to, penicillins suchas Amoxicillin, Ampicillin, Azlocillin, Carbenicillin, Cloxacillin,Dicloxacillin, Flucloxacillin, Mezlocillin, Methicillin, Nafcillin,Oxacillin, Penicillin G, Penicillin V, Piperacillin, Penicillin G,Temocillin, and Ticarcillin, and combinations of penicillins with othertherapeutic agents, such as Amoxicillin/clavulanate,Ampicillin/sulbactam, Piperacillin/tazobactam, andTicarcillin/clavulanate.

For treating infections such as tuberculosis, combination therapy mayinclude one or more of the following agents: rifampicin, rifabutin,isoniazid, streptomycin, pyrazinamide, and ethambutol. Pyridoxine(vitamin B6) (25-50 mg daily or 50-100 mg twice weekly) can beadministered to patients undergoing TB treatment with isoniazid, toreduce the occurrence of isoniazid-induced side effects in the centraland peripheral nervous system.

For individuals traveling to areas with a high incidence oftuberculosis, health care providers likely to come into contact withtuberculosis patients, or HIV-infected patients at increased risk forcatching tuberculosis, preventative therapy may be desired. Patientsundergoing preventive treatment for TB can optionally receive aperiodic, for example, a monthly clinical evaluation of their adherenceto treatment and medication side effects.

In one embodiment, the preventive therapy regimens include the use of acombination of at least two antituberculosis drugs to which theinfecting strain is believed to be susceptible (e.g., rifabutin orrifampicin, in combination with ethambutol pyrazinamide, levofloxacin orethambutol). The clinician can review the drug-susceptibility pattern ofthe M. tuberculosis strain isolated from the infecting source-patientbefore choosing a preventive therapy regimen. When combined with theformulations described herein, the patient's lungs will be at optimallevels of homeostasis, and thus less likely to become infected,particularly when a preventative antibiotic formulation isco-administered.

Drug Delivery Systems

The compositions described herein can be administered in any of avariety of drug delivery systems, depending on the intended area inwhich the compounds are to be administered. Representative drug deliverysystems include, but are not limited to, the following: eye drops,nebulizers, topical gels and ointments, dry powders, particles, sprays,liquids, anesthetic machines or vaporizers, autoinjectors, intrauterinedevices, respimats, liniments, liposomes, lotions, formulations forintramuscular, intrathecal, or subcutaneous injection, douches,infusions, and face masks. Exemplary formulations are described in moredetail below.

Formulations for Pulmonary Delivery

In some embodiments, the pharmaceutical compositions of this inventionare adapted to be administered to the lower respiratory tract (e.g., thelungs) directly through the airways by inhalation. Compositions foradministration by inhalation may take the form of inhalable powdercompositions or liquid or powder sprays, and can be administrated instandard form using powder inhaler devices or aerosol dispensingdevices. Such devices are well known. For administration by inhalation,the powdered formulations typically comprise the active compoundtogether with an inert solid powdered diluent such as lactose or starch.Inhalable dry powder compositions may be presented in capsules andcartridges of gelatin or a like material, or blisters of laminatedaluminum foil for use in an inhaler or insufflators. Each capsule orcartridge may generally contain e.g., from about 10 mg to about 100 g ofeach active compound. Alternatively, the composition of the inventionmay be presented without excipients.

The inhalable compositions may be packaged for unit dose or multi-dosedelivery. For example, the compositions can be packaged for multi-dosedelivery in a manner analogous to that described in GB 2242134, U.S.Pat. Nos. 6,632,666, 5,860,419, 5,873,360 and 5,590,645 (allillustrating the “Diskus” device), or GB2i78965, GB2129691, GB2169265,U.S. Pat. Nos. 4,778,054, 4,811,731 and 5,035,237 (which illustrate the“Diskhaler” device), or EP 69715 (“Turbuhaler” device), or GB 2064336and U.S. Pat. No. 4,353,656 (“Rotahaler” device).

Spray compositions for topical delivery to the lung by inhalation may beformulated as aqueous solutions or suspensions or as aerosols deliveredfrom pressurized packs, such as a metered dose inhaler (MDI), with theuse of a suitable liquefied propellant. The medication in pressurizedMDI is most commonly stored in solution in a pressurized canister thatcontains a propellant, although it may also be a suspension.

Aerosol compositions suitable for inhalation can be presented either assuspensions or as solutions and typically contain the active compoundand a suitable propellant such as a fluorocarbon or hydrogen-containingchlorofluorocarbon or mixtures thereof, particularly hydrofluoroalkanessuch as dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, and especially 1,1,1,2-tetrafluoroethane,1,1,1,2,3,3,3-heptafluoro-n-propane and mixtures thereof.

The aerosol composition may optionally contain additional excipientstypically associated with such compositions, for example, surfactantssuch as oleic acid or lecithin and co-solvents such as ethanol.Pressurized formulations will generally be contained within a canister(for example an aluminum canister) closed with a metering valve andfitted into an actuator provided with a mouthpiece.

Medicaments for administration by inhalation typically have a controlledparticle size. The optimum particle size for inhalation into thebronchial system is usually about 1 to about 10 μm, and in someembodiments, from about 2 to about 5 μm. Particles having a size aboveabout 20 μm are generally too large when inhaled to reach the smallairways. To achieve these particle sizes the particles of the activeingredient may be subjected to a size reducing process such asmicronization. The desired size fraction may be separated out by airclassification or sieving. Preferably, the particles will becrystalline. When an excipient such as lactose is employed, typicallythe particle size of the excipient will be much greater than theparticle size of the active ingredient.

Intranasal sprays may be formulated with aqueous or non-aqueous vehicleswith the addition of agents such as thickening agents, buffer salts oracid or alkali to adjust the pH, isotonic adjusting agents oranti-oxidants.

Solutions for inhalation by nebulization may be formulated with anaqueous vehicle with the addition of agents such as acid or alkali,buffer salts, isotonic adjusting agents or antimicrobial agents. Theymay be sterilized by filtration or heating in an autoclave, or presentedas a non-sterile product. Nebulizers supply the aerosol as a mistcreated from an aqueous formulation.

In some embodiments, the pharmaceutical compositions of this inventioncan be formulated with supplementary active ingredients. In someembodiments, the supplementary active ingredients are anti-inflammatoryagents or inhaled steroids, corticosteroids cysteinyl-leukotrienereceptor antagonist and cromolyn and or bronchodilators such as P₂agonists and/or anticholinergics. Some inhaled steroids may includeDexamethasone, Budesonide (PULMICORT®), Fluticasone (FLOVENT®),Ciclesonide (ALVESCO®), Beclomethasone Dipropionate (QVAR®) or othersknown in the art. β₂ agonists may include salbutamol, albuterol,terbutaline, salmeterol, or formoterol. An anticholinergic may includeIpratropium.

In some embodiments, the pharmaceutical composition of this invention isadministered from a dry powder inhaler.

In other embodiments, the pharmaceutical composition of this inventionis administered by an aerosol dispensing device, optionally inconjunction with an inhalation chamber such as the “Volumatic”®inhalation chamber.

The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propylene glycoland liquid polyethylene glycol, and the like), suitable mixturesthereof, and/or vegetable oils. The proper fluidity can be maintained,for example, by the use of a coating such as, for example, lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. Preventing the action of microorganismsin the compositions of the invention is achieved by adding antibacterialand/or antifungal agents, for example, parabens, chlorobutanol, phenol,sorbic acid, thimerosal and the like. In many cases, it will bepreferable to include isotonic agents, for example, sugars or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the use in the compositions of agents delayingabsorption, for example, aluminum monostearate and gelatin.

In some embodiments, a pharmaceutical composition of this invention canbe within a matrix which controls the release of the composition. Insome embodiments, the matrix can comprise lipid, polyvinyl alcohol,polyvinyl acetate, polycaprolactone, poly(glycolic)acid,poly(lactic)acid, polycaprolactone, polylactic acid, polyanhydrides,polylactide-co-glycolides, polyamino acids, polyethylene oxide, acrylicterminated polyethylene oxide, polyamides, polyethylenes,polyacrylonitriles, polyphosphazenes, poly(ortho esters), sucroseacetate isobutyrate (SAIB), and combinations thereof and other polymerssuch as those disclosed, for example, in U.S. Pat. Nos. 6,667,371;6,613,355; 6,596,296; 6,413,536; 5,968,543; 4,079,038; 4,093,709;4,131,648; 4,138,344; 4,180,646; 4,304,767; 4,946,931, each of which isexpressly incorporated by reference herein in its entirety. In theseembodiments, the matrix sustainedly releases the drug.

Pharmaceutically acceptable carriers and/or diluents may also includeany and all solvents, dispersion media, coatings, antibacterials and/orantifungals, isotonic and absorption delaying agents and the like. Theuse of such media and agents for pharmaceutically active substances iswell known in the art. Except insofar as any conventional medium oragent is incompatible with the active ingredient, use thereof in thepharmaceutical compositions is contemplated.

The pharmaceutical compositions of this invention can optionallycomprise one or more additional components, such as, but not limited to,carriers, excipients, viscosity-increasing agents, preservers,stabilizers, anti-oxidants, binders, disintegrants, humectants,lubricants, colorants, flavoring agents, corrigents, suspend moldingagents, emulsifying agents, solubilizers, buffering agents, tonicityagents, detergents, soothing agents, sulfur-containing reducing agents,etc.

The pharmaceutical compositions of the present invention can beformulated for administration in accordance with conventionaltechniques. See, e.g., Remington, The Science and Practice of Pharmacy(20th Ed. 2000). For example, the intranasal pharmaceutical compositionsof the present invention can be formulated as an aerosol (this termincludes both liquid and dry powder aerosols). Aerosols of liquidparticles can be produced by any suitable means, such as with apressure-driven aerosol nebulizer or an ultrasonic nebulizer, as isknown to those of skill in the art. See, e.g., U.S. Pat. No. 4,501,729.Aerosols of solid particles (e.g., lyophilized, freeze dried, etc.) canlikewise be produced with any solid particulate medicament aerosolgenerator, by techniques known in the pharmaceutical art. As anotherexample, the pharmaceutical compositions of the present invention can beformulated as an on-demand dissolvable form, which provides alyophilized portion of the pharmaceutical composition and a dissolvingsolution portion of the pharmaceutical composition.

In some embodiments of the present invention, the pharmaceuticalcomposition is in the form of an aqueous suspension, which can beprepared from solutions or suspensions. With respect to solutions orsuspensions, dosage forms can be comprised of micelles of lipophilicsubstances, liposomes (phospholipid vesicles/membranes) and/or a fattyacid (e.g., palmitic acid). In particular embodiments, thepharmaceutical composition is a solution or suspension that is capableof dissolving in the fluid secreted by mucous membranes of theepithelium of the tissue to which it is administered, applied and/ordelivered, which can advantageously enhance absorption.

The pharmaceutical composition can be an aqueous solution, a nonaqueoussolution or a combination of an aqueous and nonaqueous solution.

Suitable aqueous solutions include, but are not limited to, aqueousgels, aqueous suspensions, aqueous microsphere suspensions, aqueousmicrosphere dispersions, aqueous liposomal dispersions, aqueous micellesof liposomes, aqueous microemulsions, and any combination of theforegoing, or any other aqueous solution that can dissolve in the fluidsecreted by the mucosal membranes of the nasal cavity. Exemplarynonaqueous solutions include, but are not limited to, nonaqueous gels,nonaqueous suspensions, nonaqueous microsphere suspensions, nonaqueousmicrosphere dispersions, nonaqueous liposomal dispersions, nonaqueousemulsions, nonaqueous microemulsions, and any combination of theforegoing, or any other nonaqueous solution that can dissolve or mix inthe fluid secreted by mucosal membranes.

Examples of powder formulations include, without limitation, simplepowder mixtures, micronized powders, freeze dried powder, lyophilizedpowder, powder microspheres, coated powder microspheres, liposomaldispersions, and any combination of the foregoing. Powder microspherescan be formed from various polysaccharides and celluloses, which includewithout limitation starch, methylcellulose, xanthan gum,carboxymethylcellulose, hydroxypropyl cellulose, carbomer, alginatepolyvinyl alcohol, acacia, chitosans, and any combination thereof.

In particular embodiments, the composition is one that is at leastpartially, or even substantially (e.g., at least 80%, 90%, 95% or more)soluble in the fluids that are secreted by mucosa so as to facilitateabsorption. Alternatively or additionally, the composition can beformulated with a carrier and/or other substances that fosterdissolution of the agent within secretions, including without limitationfatty acids (e.g., palmitic acid), gangliosides (e.g., GM-1),phospholipids (e.g., phosphatidylserine), and emulsifiers (e.g.,polysorbate 80).

Those skilled in the art will appreciate that for intranasaladministration or delivery, because the volume of the pharmaceuticalcomposition administered is generally small, nasal secretions may alterthe pH of the administered dose since the range of pH in the nasalcavity can be as wide as 5 to 8. Such alterations can affect theconcentration of un-ionized drug available for absorption. Accordingly,in representative embodiments, the pharmaceutical composition furthercomprises a buffer to maintain or regulate pH in situ. Typical buffersinclude, but are not limited to, ascorbate, acetate, citrate, prolamine,carbonate, and phosphate buffers.

In embodiments of the invention, the pH of the pharmaceuticalcomposition is selected so that the internal environment of the mucosaltissue after administration is on the acidic to neutral side, which (1)can provide the active compound in an un-ionized form for absorption,(2) prevents growth of pathogenic bacteria, which is more likely tooccur in an alkaline environment, and (3) reduces the likelihood ofirritation of the mucosa.

For liquid and powder sprays or aerosols, the pharmaceutical compositioncan be formulated to have any suitable and desired particle or dropletsize. In illustrative embodiments, the majority and/or the mean size ofthe particles or droplets range from equal to or greater than about 1,2.5, 5, 10, 15 or 20 microns and/or equal to or less than about 25, 30,40, 45, 50, 60, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325,350, 375, 400, or 425 microns (including all combinations of theforegoing). Representative examples of suitable ranges for the majorityand/or mean particle or droplet size include, without limitation, fromabout 5 to 100 microns, from about 10 to 60 microns, from about 175 to325 microns, and from about 220 to 300 microns which facilitate thedeposition of an effective amount of the active compound, for example,in the nasal cavity (e.g., in the upper third of the nasal cavity, thesuperior meatus, the olfactory region and/or the sinus region to targetthe olfactory neural pathway). In general, particles or droplets smallerthan about 5 microns will be deposited in the trachea or even the lung,whereas particles or droplets that are about 50 microns or largergenerally do not reach the nasal cavity and are deposited in theanterior nose.

International patent publication WO 2005/023335 (Kurve Technology, Inc.)describes particles and droplets having a diameter size suitable for thepractice of representative embodiments of the present invention. Forexample, the particles or droplets can have a mean diameter of about 2to 50 microns, about 5 to 50 microns, about 5 to 40 microns, about 5 to35 microns, about 5 to 30 microns, about 5 to 20 microns, about 5 to 17microns, about 5 to 30 microns, about 10 to 25 microns, about 10 to 15microns, about 11 to 50 microns, about 11 to 30 microns, about 11 to 20microns, about 11 to 15 microns, about 12 to 17 microns, about 15 to 25microns, about 15 to 27 microns or about 17 to 23 microns.

In particular embodiments, the particles or droplets have a meandiameter of about 5 to 30 microns, about 10 to 20 microns, about 10 to17 microns, about 10 to 15 microns, about 12 to 17 microns, about 10 to15 microns or about 10 to 12 microns.

Further, the particles or droplets can have a mean diameter of about 10to 20 microns, about 10 to 25 microns, about 10 to 30 microns, or about15 to 30 microns.

The particles can “substantially” have a mean diameter or size asdescribed herein, i.e., at least about 50%, 60%, 70%, 80%, 90% or 95 ormore of the particles are of the indicated diameter or size range.

The pharmaceutical composition of this invention can be delivered as anebulized or atomized liquid having a droplet size as described above.

In some embodiments, the pharmaceutical composition is isotonic toslightly hypertonic, e.g., having an osmolarity ranging from about 150to 550 mOsM. In other embodiments, the pharmaceutical composition isisotonic, having, e.g., an osmolarity ranging from approximately 150 to350 mOsM.

According to particular embodiments of this invention that comprisemethods of intranasal delivery, it can be desirable to prolong theresidence time of the pharmaceutical composition in the nasal cavity(e.g., in the upper third of the nasal cavity, the superior meatus, theolfactory region and/or in the sinus region), for example, to enhanceabsorption. Thus, the pharmaceutical composition can optionally beformulated with a bioadhesive polymer, a gum (e.g., xanthan gum),chitosan (e.g., highly purified cationic polysaccharide), pectin (or anycarbohydrate that thickens like a gel or emulsifies when applied tonasal mucosa), a microsphere (e.g., starch, albumin, dextran,cyclodextrin), gelatin, a liposome, carbamer, polyvinyl alcohol,alginate, acacia, chitosans and/or cellulose (e.g., methyl or propyl;hydroxyl or carboxy; carboxymethyl or hydroxylpropyl), which are agentsthat enhance residence time in the nasal cavity. As a further approach,increasing the viscosity of the formulation can also provide a means ofprolonging contact of the agent with the nasal epithelium. Thepharmaceutical composition can be formulated as a nasal emulsion,ointment or gel, which offers advantages for local application becauseof their viscosity.

Moist and highly vascularized membranes can facilitate rapid absorption;consequently, the pharmaceutical composition can optionally comprise ahumectant, particularly in the case of a gel-based composition so as toassure adequate intranasal moisture content. Examples of suitablehumectants include but are not limited to glycerin or glycerol, mineraloil, vegetable oil, membrane conditioners, soothing agents, and/or sugaralcohols (e.g., xylitol, sorbitol; and/or mannitol). The concentrationof the humectant in the pharmaceutical composition will vary dependingupon the agent selected and the formulation.

The pharmaceutical composition can also optionally include an absorptionenhancer, such as an agent that inhibits enzyme activity, reduces mucousviscosity or elasticity, decreases mucociliary clearance effects, openstight junctions, and/or solubilizes the active compound. Chemicalenhancers are known in the art and include chelating agents (e.g.,EDTA), fatty acids, bile acid salts, surfactants, and/or preservatives.Enhancers for penetration can be particularly useful when formulatingcompounds that exhibit poor membrane permeability, lack oflipophilicity, and/or are degraded by aminopeptidases. The concentrationof the absorption enhancer in the pharmaceutical composition will varydepending upon the agent selected and the formulation.

To extend shelf life, preservatives can optionally be added to thepharmaceutical composition. Suitable preservatives include but are notlimited to benzyl alcohol, parabens, thimerosal, chlorobutanol andbenzalkonium chloride, and combinations of the foregoing. Theconcentration of the preservative will vary depending upon thepreservative used, the compound being formulated, the formulation, andthe like. In representative embodiments, the preservative is present inan amount of about 2% by weight or less.

The pharmaceutical compositions described herein can optionally containan odorant, e.g., as described in EP 0 504 263 B1, to provide asensation of odor, to aid in inhalation of the composition so as topromote delivery to the olfactory region and/or to trigger transport bythe olfactory neurons.

As another option, the composition can comprise a flavoring agent, e.g.,to enhance the taste and/or acceptability of the composition to thesubject.

Porous Particles for Pulmonary Administration

In one embodiment, the particles are porous, so that they have anappropriate density to avoid deposition in the back of the throat whenadministered via an inhaler. The combination of relatively largeparticle size and relatively low density avoids phagocytosis in thelungs, provides appropriately targeted delivery, avoids systemicdelivery of the components, and provides a high concentration of thecomponents in the lung.

Representative methods for preparing such particles, and for deliveringsuch particles, are described, for example, in U.S. Pat. No. 7,384,649,entitled, “Particulate compositions for pulmonary delivery,” U.S. Pat.No. 7,182,961, entitled “Particulate compositions for pulmonarydelivery,” U.S. Pat. No. 7,146,978, entitled, “Inhalation device andmethod,” U.S. Pat. No. 7,048,908, entitled “Particles for inhalationhaving sustained release properties,” U.S. Pat. No. 6,956,021, entitled“Stable spray-dried protein formulations,” U.S. Pat. No. 6,766,799,entitled “Inhalation device,” and U.S. Pat. No. 6,732,732, entitled“Inhalation device and method.”

Additional patents disclosing such particles include U.S. Pat. No.7,279,182, entitled “Formulation for spray-drying large porousparticles,” U.S. Pat. No. 7,252,840, entitled “Use of simple amino acidsto form porous particles,” U.S. Pat. No. 7,032,593, entitled “Inhalationdevice and method,” U.S. Pat. No. 7,008,644, entitled “Method andapparatus for producing dry particles,” U.S. Pat. No. 6,848,197,entitled “Control of process humidity to produce large, porousparticles,” and U.S. Pat. No. 6,749,835, entitled “Formulation forspray-drying large porous particles.”

U.S. Pat. No. 7,678,364, entitled “Particles for inhalation havingsustained release properties,” discloses methods for deliveringparticles to the pulmonary system comprising: administering to therespiratory tract of a patient in need of treatment, prophylaxis ordiagnosis an effective amount of a dry powder comprising: a) amultivalent metal cation which is complexed with a therapeutic,prophylactic or diagnostic agent; b) a pharmaceutically acceptablecarrier; and c) a multivalent metal cation-containing component whereinthe dry powder is spray-dried and has a total amount of multivalentmetal cation which is about 10% w/w or more of the total weight of theagent, a tap density of about 0.4 g/cm³ or less, a median geometricdiameter of from about 5 micrometers to about 30 micrometers and anaerodynamic diameter of from about 1 to about 5 microns.

Delivery of bioactive agents to the pulmonary system typically resultsin rapid release of the agent following administration. For example,Further, Heinemann, Traut and Heise teach in Diabetic Medicine 14:63-72(1997) that the onset of action, assessed by glucose infusion rate, inhealthy volunteers after inhalation was rapid with the half-maximalaction reached in about 30 minutes. That said, the formulation can beprepared so that the components are released into the lungs in asustained fashion.

Particles suitable for inhalation can be designed to possess a sustainedrelease profile. This sustained released profile provides for prolongedresidence of the administered components in the lung and thereby,increases the amount of time in which therapeutic levels of thecomponents are present in the local environment. Consequently, patientcompliance and comfort can be increased by not only reducing frequencyof dosing, but by providing a therapy which is more amenable andefficacious to patients.

The particles comprise a pharmaceutically acceptable carrier. Suitablepharmaceutically acceptable carriers can be chosen, for example, basedon achieving particles having the proper characteristics for inhalationto the area of the respiratory tract where delivery is desired andtherapeutic action is achieved.

In a preferred embodiment of the invention, the pharmaceuticallyacceptable carrier is a phospholipid. The phospholipid can be present inthe particles in an amount ranging from about 0 to about 90 weight %.More commonly it can be present in the particles in an amount rangingfrom about 10 to about 60 weight %. Examples of suitable phospholipidsinclude, among others, phosphatidic acids, phosphatidylcholines,phosphatidylalkanolamines such as a phosphatidylethanolamines,phosphatidylglycerols, phosphatidylserines, phosphatidylinositols andcombinations thereof. Other phospholipids are known to those skilled inthe art and are described in U.S. patent application Ser. No. 09/752,109entitled “Particles for Inhalation Having Sustained Release Properties”filed on Dec. 29, 2000 and U.S. patent application Ser. No. 09/752,106entitled “Particles for Inhalation Having Sustained release Properties”filed on Dec. 29, 2000 the contents of all of which are incorporatedherein in their entirety. In a preferred embodiment, the phospholipidsare endogenous to the lung.

The phospholipids can be selected to impart controlled releaseproperties to the highly dispersible particles. The phase transitiontemperature of a specific phospholipid can be below, around or above thephysiological body temperature of a patient. Preferred phase transitiontemperatures range from 30 to 50° C., (e.g., within +/−10 degrees of thenormal body temperature of patient). By selecting phospholipids orcombinations of phospholipids according to their phase transitiontemperature, the particles can be tailored to have controlled releaseproperties. For example, by administering particles which include aphospholipid or combination of phospholipids which have a phasetransition temperature higher than the patient's body temperature, therelease of active agent can be slowed down. On the other hand, rapidrelease can be obtained by including in the particles phospholipidshaving lower transition temperatures. Particles having controlledrelease properties and methods of modulating release of a biologicallyactive agent are described in U.S. patent application Ser. No.09/644,736 entitled Modulation of Release From Dry Powder Formulationsby Controlling Matrix Transition, filed on Aug. 23, 2000, the entirecontents of which are incorporated herein by reference.

The amount of the glutathione, ascorbic acid or salt thereof, and sodiumbicarbonate present in the particles can range from about 0.1 weight %to about 95 weight %, though in some cases, can even be as high as 100%.For example, from about 1 to about 50%, such as from about 5 to about30%. Particles in which the drug is distributed throughout a particlecan be preferred.

In a further embodiment, the particles can also include other excipientssuch as, for example, buffer salts, dextran, polysaccharides, lactose,trehalose, cyclodextrins, proteins, polycationic complexing agents,peptides, polypeptides, fatty acids, fatty acid esters, inorganiccompounds, phosphates.

In one embodiment, the particles can further comprise polymers.Biocompatible or biodegradable polymers are preferred. Such polymers aredescribed, for example, in U.S. Pat. No. 5,874,064, issued on Feb. 23,1999 to Edwards et al., the teachings of which are incorporated hereinby reference in their entirety.

In yet another embodiment, the particles include a surfactant other thanthe phospholipids described above. As used herein, the term “surfactant”refers to any agent which preferentially absorbs to an interface betweentwo immiscible phases, such as the interface between water and anorganic polymer solution, a water/air interface or organic solvent/airinterface. Surfactants generally possess a hydrophilic moiety and alipophilic moiety, such that, upon absorbing to particles, they tend topresent moieties to the external environment that do not attractsimilarly-coated particles, thus reducing particle agglomeration.Surfactants may also promote absorption of a therapeutic or diagnosticagent and increase bioavailability of the agent.

Suitable surfactants which can be employed in fabricating the particlesof the invention include but are not limited to hexadecanol; fattyalcohols such as polyethylene glycol (PEG); polyoxyethylene-9-laurylether; a surface active fatty acid, such as palmitic acid or oleic acid;glycocholate; surfactin; a poloxamer; a sorbitan fatty acid ester suchas sorbitan trioleate (Span 85); Tween 80 and tyloxapol.

The surfactant can be present in the particles in an amount ranging fromabout 0 to about 5 weight %. Preferably, it can be present in theparticles in an amount ranging from about 0.1 to about 1.0 weight %.

The particles, also referred to herein as powder, can be in the form ofa dry powder suitable for inhalation. In a particular embodiment, theparticles can have a tap density of less than about 0.4 g/cm³. Particleswhich have a tap density of less than about 0.4 g/cm³ are referred toherein as “aerodynamically light particles.” More preferred areparticles having a tap density less than about 0.1 g/cm³.

Aerodynamically light particles have a preferred size, e.g., a volumemedian geometric diameter (VMGD) of at least about 5 microns (μm). Inone embodiment, the VMGD is from about 5 μm to about 30 μm. In anotherembodiment of the invention, the particles have a VMGD ranging fromabout 9 μm to about 30 μm. In other embodiments, the particles have amedian diameter, mass median diameter (MMD), a mass median envelopediameter (MMED) or a mass median geometric diameter (MMGD) of at least 5μm, for example from about 5 μm to about 30 μm.

Aerodynamically light particles preferably have “mass median aerodynamicdiameter” (MMAD), also referred to herein as “aerodynamic diameter,”from about 1 μm to about 5 μm. In one embodiment of the invention, theMMAD is from about 1 μm to about 3 μm. In another embodiment, the MMADis from about 3 μm to about 5 μM.

In another embodiment of the invention, the particles have an envelopemass density, also referred to herein as “mass density” of less thanabout 0.4 g/cm³. The envelope mass density of an isotropic particle isdefined as the mass of the particle divided by the minimum sphereenvelope volume within which it can be enclosed.

Tap density can be measured by using instruments known to those skilledin the art such as the Dual Platform Microprocessor Controlled TapDensity Tester (Vankel, N.C.) or a GeoPyc™ instrument (MicrometricsInstrument Corp., Norcross, Ga. 30093). Tap density is a standardmeasure of the envelope mass density. Tap density can be determinedusing the method of USP Bulk Density and Tapped Density, United StatesPharmacopia convention, Rockville, Md., 10^(th) Supplement, 4950-4951,1999. Features which can contribute to low tap density include irregularsurface texture and porous structure.

The diameter of the particles, for example, their VMGD, can be measuredusing an electrical zone sensing instrument such as a Multisizer Ile,(Coulter Electronic, Luton, Beds, England), or a laser diffractioninstrument (for example Helos, manufactured by Sympatec, Princeton,N.J.). Other instruments for measuring particle diameter are well knownin the art. The diameter of particles in a sample will range dependingupon factors such as particle composition and methods of synthesis. Thedistribution of size of particles in a sample can be selected to permitoptimal deposition within targeted sites within the respiratory tract.

Particles that have a tap density less than about 0.4 g/cm³, mediandiameters of at least about 5 μm, and an aerodynamic diameter of fromabout 1 μm to about 5 μm, or from about 1 μm to about 3 μm, are morecapable of escaping inertial and gravitational deposition in theoropharyngeal region, and are targeted to the airways or the deep lung.The use of larger, more porous particles is advantageous since they areable to aerosolize more efficiently than smaller, denser aerosolparticles such as those currently used for inhalation therapies.

In comparison to smaller particles, the larger aerodynamically lightparticles, preferably having a VMGD of at least about 5 μm, also canpotentially more successfully avoid phagocytic engulfment by alveolarmacrophages and clearance from the lungs, due to size exclusion of theparticles from the phagocytes' cytosolic space. Phagocytosis ofparticles by alveolar macrophages diminishes precipitously as particlediameter increases beyond about 3 μm. Kawaguchi, H., et al, Biomaterials7:61-66 (1986); Krenis, L. J. and Strauss, B., Proc. Soc. Exp. Med.,107:748-750 (1961); and Rudt, S, and Muller, R. H., J Contr. Rel.,22:263-272 (1992). For particles of statistically isotropic shape, suchas spheres with rough surfaces, the particle envelope volume isapproximately equivalent to the volume of cytosolic space requiredwithin a macrophage for complete particle phagocytosis.

The particles can be prepared, for example, by spray drying. Forexample, a spray drying mixture, also referred to herein as “feedsolution” or “feed mixture,” which includes the therapeutic agentsdescribed herein, a pharmaceutically acceptable carrier, and optionallya multivalent metal cation component are fed to a spray dryer.

The total amount of solvent or solvents being employed in the mixturebeing spray dried generally is greater than 99 weight percent. Theamount of solids (drug, charged lipid and other ingredients) present inthe mixture being spray dried generally is less than about 1.0 weightpercent. Preferably, the amount of solids in the mixture being spraydried ranges from about 0.05% to about 0.5% by weight.

Using a mixture which includes an organic and an aqueous solvent in thespray drying process allows for the combination of hydrophilic andhydrophobic components, while not requiring the formation of liposomesor other structures or complexes to facilitate solubilization of thecombination of such components within the particles.

Suitable spray-drying techniques are described, for example, by K.Masters in “Spray Drying Handbook,” John Wiley & Sons, New York, 1984.Generally, during spray-drying, heat from a hot gas such as heated airor nitrogen is used to evaporate the solvent from droplets formed byatomizing a continuous liquid feed. Other spray-drying techniques arewell known to those skilled in the art. In a preferred embodiment, arotary atomizer is employed. An example of a suitable spray dryer usingrotary atomization includes the Mobile Minor spray dryer, manufacturedby Niro, Inc., Denmark. The hot gas can be, for example, air, nitrogenor argon.

Preferably, the particles are obtained by spray drying using an inlettemperature from about 100 to about 400° C. and an outlet temperaturefrom about 50 to about 130° C.

The spray dried particles can be fabricated with a rough surface textureto reduce particle agglomeration and improve flowability of the powder.The spray-dried particle can be fabricated with features which enhanceaerosolization via dry powder inhaler devices, and lead to lowerdeposition in the mouth, throat and inhaler device.

Aerosol dosage, formulations and delivery systems also may be selectedfor a particular therapeutic application, as described, for example, inGonda, I. “Aerosols for delivery of therapeutic and diagnostic agents tothe respiratory tract,” in Critical Reviews in Therapeutic Drug CarrierSystems, 6:273-313, 1990; and in Moren, “Aerosol dosage forms andformulations,” in: Aerosols in Medicine, Principles, Diagnosis andTherapy, Moren, et al., Eds, Esevier, Amsterdam, 1985.

The compounds in the formulation may be contained in any appropriateamount in any suitable carrier substance, and are generally present inan amount of 1-95% by weight of the total weight of the particles thatare administered. The pharmaceutical compositions used to deliver theindividual components (glutathione, ascorbic acid or salts thereof, andsodium bicarbonate) can be formulated to release the components at apredetermined time period by using biodegradable polymers, or otherpolymeric drug delivery systems.

When controlled release formulations are used, they are preferably a)formulations that after a predetermined lag time create a substantiallyconstant concentration of the drug within the lungs over an extendedperiod of time, or b) formulations that localize drug action by, e.g.,spatial placement of a controlled release composition adjacent to or inthe lungs, for example, using mucoadhesive polymers.

Examples of suitable polymeric materials include acrylic polymers,methacrylic acid copolymers with an acrylic or methacrylic ester (e.g.,methacrylic acid ethylacrylate copolymer (1:1) and methacrylic acidmethylmethacrylate copolymer (1:2), polyvinyl acetate phthalate,hydroxypropyl cellulose acetate phthalate and cellulose acetatephthalate), as well as cellulose acetate phthalate, hydroxypropylmethylcellulose phthalate, polyvinyl acetate phthalate. Commerciallyavailable products include, for example, KOLLIKOAT®, EDRAGIT® (e.g.,EUDRAGIT 40), AQUATERIC®, AQOAT®. The enteric polymers used can also bemodified by mixing with other coating products that are not pHsensitive. Examples of such coating products include, for example, theneutral methacrylic acid esters with a small portion oftrimethylammonioethyl methacrylate chloride, sold currently under thetrade names EUDRAGIT® and EUDRA GIT® RL; a neutral ester dispersionwithout any functional groups, sold under the trade names EUDRAGIT®NE30D and EUDRAGIT®NE30, EUDRAGIT® 40; polysaccharides, like amylose,chitosan, chondroitin sulfate, dextran, guar gum, inulin and pectin; andother pH independent coating products.

The polymer in various embodiments is from about 5% to about 75% of theweight of the microgranule. In other embodiments, the polymer is fromabout 10% to about 60%, from about 20% to about 55%, from about 30% toabout 80%, or from about 25% to about 50% of the weight of themicrogranule. The weight percent of the polymer to the weight of themicrogranule can depend, in part, on the polymer used, and thetemperature of the polymer.

The microgranules may further comprise one or more of diluents,plasticizers, anti-agglomeratives, anti-sticking, glidants, anti-foamsurfactants, or coloring substances. These, along with other polymersand coatings (e.g., protective coatings, over-coatings, and films) aremore fully described below.

In order to deliver to the deep lung, it can be important to minimizeparticle agglomerization, and for this reason, excipients withanti-agglomerative properties can also be used. Examples include talc;plasticizing materials, like acetylated glycerides, diethylphthalate,propylene glycol and polyethylene glycol; surfactants like polysorbateand polyoxyethylenate esthers, anti-foaming agents, as well asanti-sticking agents.

Suitable ingredients can be incorporated into the coating formula suchas plasticizers, which include, for example, adipates, azelates,benzoates, citrates, isoebucates, phthalates, sebacates, stearates andglycols. Representative plasticizers include acetylated monoglycerides,butyl phthalyl butyl glycolate, dibutyl tartrate, diethyl phthalate,dimethyl phthalate, ethyl phthalyl ethyl glycolate, glycerin, ethyleneglycol, propylene glycol, triacetin citrate, triacetin, tripropinoin,diacetin, dibutyl phthalate, acetyl monoglyceride, polyethylene glycols,castor oil, triethyl citrate, polyhydric alcohols, acetate esters,gylcerol triacetate, acetyl triethyl citrate, dibenzyl phthalate,dihexyl phthalate, butyl octyl phthalate, diisononyl phthalate, butyloctyl phthalate, dioctyl azelate, epoxydized tallate, triisoctyltrimellitate, diethylhexyl phthalate, di-n-octyl phthalate, di-1-octylphthalate, di-1-decyl phthalate, di-n-undecyl phthalate, di-n-tridecylphthalate, tri-2-ethylhexyl trimellitate, di-2-ethylhexyl adipate,di-2-ethylhexyl sebacate, di-2-ethylhexyl azelate, dibutyl sebacate,glyceryl monocaprylate, and glyceryl monocaprate. Other various layers,as recognized by one of skill in the art are also envisioned. The amountof plasticizer used in the polymeric material typically ranges fromabout 10% to about 50%, for example, about 10, 20, 30, 40, or 50%, basedon the weight of the dry polymer. Optional modifying components of aprotective layer which can be used over the enteric or other coatingsinclude a water penetration barrier layer (semi-permeable polymer) whichcan be successively coated after the enteric or other coating to reducethe water penetration rate through the enteric coating layer and thusincrease the lag time of the drug release. Coatings commonly known toone skilled in the art can be used for this purpose by coatingtechniques such as fluid bed coating using solutions of polymers inwater or suitable organic solvents or by using aqueous polymerdispersions. For example, useful materials include cellulose acetate,cellulose acetate butyrate, cellulose acetate propionate, ethylcellulose, fatty acids and their esters, waxes, zein, and aqueouspolymer dispersions such as EUDRAGIT® RS and RL 3OD, EUDRAGIT® NE 3OD,EUDRAGIT® 40, AQUACOAT®, SURELEASE®, cellulose acetate latex.Combinations of the polymers and hydrophilic polymers such as hydroxyethyl cellulose, hydroxypropyl cellulose (KLUCEL®, Hercules Corp.),hydroxypropyl methylcellulose (METHOCEL®, Dow Chemical Corp.),polyvinylpyrrolidone may also be used.

The amount of polymer to be used in the formulations is typicallyadjusted to achieve the desired drug delivery properties, including theamount of drug to be delivered, the rate and location of drug delivery,the time delay of drug release, and the size of the multiparticulates inthe formulation. The combination of all solid components of thepolymeric material, including co-polymers, fillers, plasticizers, andoptional excipients and processing aids, typically provides about 1% toabout 50% weight of the core.

Colon Targeted Drug Delivery Systems

Local delivery to the colon allows topical treatment of colonicdisorders such as ulcerative colitis, inflammatory bowel disease,Crohn's disease, amebiosis, colonic cancer, local treatment of colonicpathologies, and systemic delivery of protein and peptide drugs Crohn'sdisease, and the like. There are a number of approaches for CDDS (ColonSpecific Drug Delivery), including pH and time dependent systems,microbially-triggered systems, and pressure-controlled colonic deliverycapsules, CODES™, and osmotic controlled drug delivery.

The colon specific drug delivery system (CDDS) should prohibitsignificant drug release and absorption in the stomach and the smallintestine, predominantly only release the therapeutic agents once thesystem reaches the colon. It is preferred to administer the drugdelivery systems via the oral route. Rectal administration offers theshortest route for targeting therapeutic agents to the colon, but it isdifficult to reach the proximal part of colon via rectal administration.Drug preparation for intrarectal administration is typically in the formof solutions, foam, and suppositories.

Chronic colitis, namely ulcerative colitis, and Crohn's disease arecurrently treated with glucocorticoids, such as dexamethasone and methylprednisolone, and other anti-inflammatory agents. Glucocorticoids cancause systemic side effects, including adenosuppression,immunosuppression, cushinoid symptoms, and bone resorption, when givensystemically. Selective delivery of these drugs to the colon, forexample, using the compositions described herein, which establish and/ormaintain homeostasis in the colonic mucosa, can both lower the requireddose and also reduce systemic side effects.

With these formulations, the compositions used to restore homeostasis tothe intestinal mucosa. Therapeutic agents can be added, includingprobiotics, 5-amino-salicylic acid and other anti-inflammatory agents,antibiotics, antivirals, and the like.

Several of the conventional approaches for site-specific drug deliveryto the colon are discussed in detail below.

pH Sensitive Polymer-Coated Drug Delivery

In the stomach, the pH ranges from 1 and 2 during fasting, but increasesafter eating. The pH is about 6.5 in the proximal small intestine, andabout 7.5 in the distal small intestine. From the ileum to the colon, pHdeclines significantly. It is about 6.4 in the cecum. However, pH valuesas low as 5.7 have been measured in the ascending colon in healthyvolunteers. The pH in the transverse colon is 6.6 and 7.0 in thedescending colon. Use of pH dependent polymers is based on thesedifferences in pH levels. The polymers described as pH dependent incolon specific drug delivery are insoluble at low pH levels but becomeincreasingly soluble as pH rises, pH-dependent polymers can protect aformulation in the stomach and proximal small intestine, but can startto dissolve in the lower small intestine, which can result in poorsite-specificity.

Delayed (Time Controlled Release System) Release Drug Delivery

Time controlled release system (TCRS), such as sustained or delayedrelease dosage forms, can also be used.

Appropriate integration of pH sensitive and time release functions intoa single dosage form can improve the site specificity of drug deliveryto the colon.

In the stomach, drug release can be suppressed using a pH-sensingfunction (acid resistance) in the dosage form. Typical enteric coatedtime-release tablets include at least three components, a drugcontaining core tablet (rapid release function), a swellable hydrophobicpolymer coating layer, such as a hydroxy propyl cellulose layer (HPC)layer, which provides a time-release function, and an enteric coatinglayer (which typically functions by being resistant to stomach acid. Thetablet does not release the drug in the stomach, by virtue of the acidresistance of the outer enteric coating layer. After gastric emptying,the enteric coating layer rapidly dissolves and the intestinal fluidbegins to slowly erode the press coated polymer (HPC) layer. After thetablet has eroded to the point where the core tablet is reached, rapiddrug release typically occurs, unless the drug itself is mixed with adegradable polymer, which can provide sustained release dependent on thedegradation properties of the polymer.

Microbially-Triggered Drug Delivery

The microflora of the colon include predominantly anaerobic bacteria,e.g. Bacteroides, bifidobacteria, eubacteria, clostridia, enterococci,enterobacteria and ruminococcus. This microflora meets its energy needsby fermenting various types of undigested substrates, using enzymes likepectinase, glucoronidase, xylosidase, arabinosidase, galactosidase,nitroreductase, azareducatase, deaminase, and urea dehydroxylase.Because of the presence of the biodegradable enzymes only in the colon,the use of biodegradable polymers for colon-specific drug delivery seemsto be a more site-specific approach as compared to other approaches.Drug delivery can therefore be achieved by using materials that degradein the presence of these enzymes.

Polymers can be used as drug carriers for drug delivery to the colon,and both synthetic and naturally-occurring polymers have been used forthis purpose. Various azo polymers have been used as coating materialsover drug cores, and these are susceptible to cleavage by theazoreducatase in the large bowel. In one embodiment, the coatingincludes polymers cross-linked with azoaromatic groups, which protectthe drug from digestion in the stomach and small intestine. In thecolon, the azo bonds are reduced, and the drug is released.

Naturally-occurring polysaccharides can be used to target the colon,since polymers of such monosaccharides are found in abundance, have wideavailability, are inexpensive, and are available in a variety ofstructures with varied properties. They can be easily modifiedchemically, biochemically, and are highly stable, safe, nontoxic,hydrophilic and gel forming and in addition, are biodegradable. Theseinclude naturally occurring polysaccharides obtained from plant (guargum, inulin), animal (chitosan, chondroitin sulphate), algal (alginates)or microbial (dextran) origin. The polysaccharides can be broken down bythe colonic microflora to simple saccharides.

Pressure Controlled Drug-Delivery Systems

As a result of peristalsis, higher pressures are encountered in thecolon than in the small intestine. Takaya et al, developed pressurecontrolled colon-delivery capsules prepared using ethylcellulose, whichis insoluble in water (Takaya et al., “Importance of dissolution processon systemic availability of drugs delivered by colon delivery system,”J. Control. Rel. 50:(1-3):111-122 (1998)). Drug release occurs pressurein the lumen of the colon causes the water-insoluble polymer capsule todisintegrate. In pressure controlled ethylcellulose single unitcapsules, the components are delivered in solution rather than in solidform, as would be the case with tablets.

Colon Targeted Delivery System (CODES™)

CODES™ was designed to address limitations associated with pH ortime-dependent systems (see, for example, U.S. Pat. No. 6,368,629 toWatanabe et al. and Takemura et al., Pro. Int. Sym. Control Rel. Bioact.Mat, 27 (2000)). CODES™ is a combined approach of pH-dependent andmicrobially-triggered CDDS, using lactulose as a trigger for sitespecific drug release in the colon. The system includes a traditionaltablet core comprising lactulose, which is over coated with an acidsoluble material, such as Eudragit E, and then subsequently overcoatedwith an enteric material, such as Eudragit L. The enteric coatingprotects the tablet while it is located in the stomach, but thendissolves quickly following gastric emptying. The acid soluble materialcoating then protects the preparation as it passes through the alkalinepH of the small intestine, and once the tablet arrives in the colon,bacteria enzymatically degrade the polysaccharide (lactulose) intoorganic acid. The thus-formed acid lowers the pH surrounding the systemsufficient to dissolve the acid soluble coating and release thetherapeutic agents.

Osmotic Controlled Drug Delivery (ORDS-CT)

The OROS-CT (Alza Corporation) can be used to target therapeutic agentsto the colon. The OROS-CT system includes from 1 to 6 osmotic(push-pull) units, each 4 mm in diameter, encapsulated within a hardgelatin capsule. Each bilayer push-pull unit contains an osmotic pushlayer and a drug layer, both surrounded by a semi-permeable membrane. Anorifice is drilled through the membrane next to the drug layer.Immediately after the OROS-CT is swallowed, the gelatin capsulecontaining the push-pull units dissolves. The drug-impermeable entericcoating prevents each push-pull unit from absorbing water in the acidicaqueous environment of the stomach, but as the unit enters the smallintestine, the coating dissolves. Water then enters the unit, causingthe osmotic push compartment to swell, and forming a flowable gel in thedrug compartment. The swelling forces the gel out of the orifice at aprecisely controlled rate through the semipermeable membrane. Drugrelease typically begins when the unit reaches the colon. OROS-CT unitscan be tailored to maintain a constant release rate, in the colon, forup to 24 hours, or over a period as short as four hours. Any of theseformulations can be used to administer the therapeutic agents describedherein to the colon.

Delivery to the Oral Mucosa

The formulations described herein can also be used to deliver thetherapeutic agents to the oral mucosa. The formulations can be used toestablish and maintain homeostasis in the oral mucosa, which can occurin patients with “dry mouth.” Chronic dry mouth, or xerostomia, is acommon problem that can affect about 25% of all adults. Dry mouthsymptoms can be triggered by medications, diabetes, Sjögren's Syndrome,and a variety of other causes. Symptoms of dry mouth include bad breath,a sticky, dry or sore mouth, cracking at the corners of the mouth, a redand parched mouth, blisters and mouth ulcers, and waking up with a drymouth at night.

The formulations, which include glutathione, ascorbic acid and/or itssalts, and sodium bicarbonate, can alleviate dry mouth. Accordingly,they can be added to mouthwashes, toothpastes, gels, and the like, aswell as buccal formulations, particularly when therapeutic agents areused.

One type of user that may benefit from the formulations described hereinis a consumer of nicotine gums, such as Nicorette®. Nicotine gum cancause a sore throat in some users, can cause jaw pain and/or toothdisorders, and dry mouth occurs in approximately 6 percent of users ofsome nicotine-containing gums.

Where patients are suffering from bacterial disorders in the mouth, suchas periodontal disease, the formulations can also include, or beco-administered with, appropriate antibiotics, such as doxycycline andchlorhexidine. When patients are suffering from viral disorders such ascold sores, shingles, aphthous ulcers, and the like, the compositionscan also include, or be co-administered with, antiviral agents.Particularly when the patients suffer from pain in their mouth, whetherfrom bacterial or viral causes, physical injuries, or oral surgery,including tonsillectomies, uvulaplasties, scaling and root planning,grafting, and the like, the formulations described herein can helpmaintain homeostasis in the oral mucosa, which can accelerate healing,and can include anesthetics such as lidocaine, marcaine, xylocaine, andthe like, to help alleviate pain. Anti-inflammatory agents can also bepresent.

Drugs can also be administered using buccal drug delivery vehicles,whether to treat the above-listed indications, or to administer drugssystemically. In addition to the well-known dissolvable films, which caninclude the therapeutic agents described herein, buccal drug deliveryforms can also be used. The presence of the active agents (glutathione,ascorbic acid and its salts, and sodium bicarbonate) can help minimizeirritation to the mouth which might otherwise be caused by some drugs,and which might otherwise minimize the ability to administer such drugsvia the buccal route.

One form of buccal drug delivery uses fast melt technology, and ischaracterized by rapid drug release. A known fast melt product is Zydis,which is formulated as a wafer having a very low density and minimalquantities of excipient. Thus, for example U.S. Pat. No. 5,939,091describes a method of making fast melt tablets comprising SorbitolInstant. Similarly, WO 02/085119 describes a dosage form for intra-oraldelivery of nicotine comprising a hydroxypropylmethylcellulose film.This delivery system is characterized by rapid dissolution providing foralmost instantaneous delivery of the nicotine.

In other forms, the structure of the tablet is modified so as to providea desired delivery profile. For example, PCT WO 03/039518 describes anoral dosage formulation for delivery of nicotine comprising two layers,the first providing for buccal drug delivery and the second providingfor delivery via the stomach or intestines. This formulation provides aninitial rapid release of nicotine in the mouth followed by a slowsustained release of nicotine in the gut. PCT WO 01/37814 whichdescribes bilayered buccal tablets comprising nicotine. These tabletsprovide a biphasic release of nicotine from a modified lactose andmagnesium stearate containing tablet.

Other buccal formulations are in the form of a gum or lozenge. Forexample, PCT WO 02/076211 describes an oral dosage formulationcomprising nicotine, including a hard lozenge having a matrix which isin a glassy, i.e. amorphous physical state. These lozenges comprise asodium carbonate buffer. EP 0 500 658 describes a nicotine containingstimulant unit for buccal drug delivery, which can comprise a gumcomponent, and U.S. Pat. No. 6,183,775 describes a controlled-releaselozenge comprising soluble filler, an insoluble film-forming agent, anda swellable polymer. The lozenges are produced by compressing a drygranulate.

Other known dosage forms include troches. For example, U.S. Pat. No.3,590,111 describes troches, formed by wet and dry granulationprocedures, which include guar gum, disaccharides and hexahydricsaturated aliphatic alcohols. U.S. Pat. No. 4,829,056 discloses a buccaltablet containing at least one monosaccharide or disaccharide and locustbean gum. U.S. Pat. No. 5,470,566 discloses a chewing gum comprising agum base and a “taste enhancer,” which can include a sugar or sugaralcohol. Additional sweetening agents can also be used.

Accordingly, oral and buccal formulations can be used to provide aninitial burst release (i.e., when dissolvable films are used), orsustained release (i.e., when lozenges or other such forms are used).The release profile of the active agent or the dissolution profile ofthe lozenge is governed by the matrix composition and lozenge size, andcan be varied according to the nature of the active agent and thedesired effect. Thus, the dissolution profile can be altered, whileretaining the same amount of the active agent, by varying the lozengesize and/or the proportion of gum in the lozenge. A smaller overalllozenge size will result in faster dissolution. Similarly, a reduced gumcontent will result in faster lozenge dissolution.

A suitable dissolution profile for lozenges of the invention is suchthat after 20 minutes approximately 35-65% of the lozenge has dissolved,after 40 minutes, approximately 60-90% of the lozenge has dissolved, andafter 60 minutes more than 70% of the lozenge has dissolved. Typicallylozenges are not smaller than 300-400 mg, and are not larger thanapproximately 1.5 g, approximately 1.75 g or approximately 2 g. Ingeneral, lozenge size (in terms of dimensions and shape) should besuitable for parking the lozenge in the buccal cavity.

Any suitable gum may be used to prepare lozenges. Suitable gums includegum acacia, gum arabic, carob gum, carrageenan, ghattii gum, guar gum,karaya gum, pectin, tragacanth gum, locust bean gum and xanthan gum. Apreferred gum component is gum acacia, especially supplied in spraydried form for manufacture of lozenges.

In addition to the gum, lozenges also typically include one or morenon-crystallizing sugars and/or one or more non-crystallizing sugaralcohols. Non-crystallizing forms of sugars or sugar alcohols arecommercially available and may conveniently be used. Alternatively,sugars or sugar alcohols can be heat treated to providenon-crystallizing properties. Suitable sugars and sugar alcohols includenon-crystallizing or treated forms of dextrose, maltose, sucrose,fructose, glucose syrup, invert sugar syrup, honey, laevulose, sorbitol,xylitol, maltitol, mannitol and isomalt. Preferred non-crystallizingsugars or non-crystallizing sugar alcohols include non-crystallizingforms (or mixtures) of sorbitol, xylitol, maltitol, mannitol, andisomalt.

Lozenges will normally contain water, for example, a water content ofapproximately 5-20% by weight, and can also include appropriate buffersto maintain a pH suitable for buccal absorption of the active agent. Thebuffer can vary according to the active agent, and generally varies soas to provide a pH at which the active agent is in an non-ionized form.For example, if nicotine is present, the pH is preferably in the range7.5-9.0, more preferably 8.0-8.4. Control of the lozenge pH, and inparticular the use of phosphate buffers, can also provide an improvedtaste or “mouth feel.”

Lozenges and other oral or buccal formulations can optionally compriseflavorings, vitamins, anti-oxidants, anti-fungals, anti-bacterials,taste masking agents, colorings, excipients, stabilizers and sweeteners.Suitable components may be selected from those known in the art.

Drugs particularly suitable for delivery via the oral or buccal routeinclude alkaloids, for example nicotine, alkaloidal drugs, anti-emetics(for example 5-HT antagonists), agents for migraine treatment (forexample 5-HT agonists), analgesics (for example cannabis, .DELTA.9-THCand alkaloids), drugs that benefit from rapid uptake, drugs used inacute therapy, drugs that need to be or are preferentially taken lyingdown, drugs taken by patients who cannot or do not wish to swallow ordrugs to be taken where it is undesirable to use a large amount ofwater. Drugs are preferably readily absorbable across the buccal mucosa.Drugs particularly suitable for delivery via lozenges of the inventionare drugs for which the first pass effect is not beneficial, i.e. drugsof which the potency is reduced as a result of metabolism in the liver.Mucosal delivery is ideal for such drugs as they are directly absorbedinto the bloodstream without first passing through the liver.

Particularly preferred drugs for delivery using lozenges of theinvention include nicotine, the analgesic Δ9-THC, the anti-emeticondansetron (a 5-HT3 antagonist), and the anti-migraine drug sumatriptan(a 5-HT1 agonist). Drugs for delivery using lozenges of the inventionmay optionally be in the form of a pharmaceutically acceptable salt.

The oral or buccal drug delivery systems can be used to administer anysuitable dose of an active agent. Typical doses may be in the range0.5-10 mg, but doses of approximately up to 200 mg can be delivered.

The use of lozenges is particularly suitable for active agents where itis desirable to limit patient exposure to the agent. The controlledrelease characteristics of the lozenges allow self-titration of the drugdosage by the patient. This is useful, for example, when the lozengesare used to deliver agents for migraine treatment or analgesics. Oncesufficient active agent has been absorbed to overcome the symptoms forwhich the agent has been administered, the remainder of the lozenge canbe removed from the patient's mouth.

Vaginal and Rectal Formulations

When used to treat or prevent disorders in the rectum or vagina, theformulations include the components described herein (i.e., glutathioneor a salt, prodrug, or derivative thereof, an organic acid or a saltthereof, bicarbonate, and the like), as well as conventional lubricantsused in such formulations.

Composition for rectal and/or vaginal administration can be formulatedfor topical administration, and in certain embodiments the compositionis formulated as a gel, or formulated as a topical cream, ointment,lotion or foam formulation. The composition can further comprise apharmaceutically acceptable excipient, a lubricant, an antifungal,antibacterial, or antiviral agent, an antipruritic agent, or ananesthetic, for example.

Other therapeutic agents used to treat disorders of the rectum and/orvagina can be added, as appropriate.

The formulations suitable for vaginal or rectal administration can bepresent as aqueous or oily suspensions, solutions or emulsions (liquidformulations) containing in addition to the active ingredients, suchcarriers as are known in the art to be appropriate. For “stand-alone”lubricants (i.e., lubricants that are not pre-packaged with condoms),gels and similar aqueous formulations are generally preferred, forvarious reasons known to those skilled in the art.

The topical formulations can be applied to the vagina and/or the anus.The composition administered to the anus is suitably a foam or gel,etc., such as those described above with regard to vaginal application.In the case of anal application, it may be preferred to use anapplicator which distributes the composition substantially evenlythroughout the anus. For example, a suitable applicator is a tube 2.5 to25 cm, preferably 5 to 10 cm, in length having holes distributedregularly along its length.

When the composition is a water-soluble vaginal cream or gel, suitably0.1 to 4 grams, preferably about 0.5 to 2 grams, are applied. When thecomposition is a vaginal spray-foam, suitably 0.1 to 2 grams, preferablyabout 0.5 to 1 grams, of the spray-foam are applied. When thecomposition is an anal cream or gel, suitably 0.1 to 4 grams, preferablyabout 0.5 to 2 grams of the cream or gel is applied. When thecomposition is an anal spray-foam, suitably 0.1 to 2 grams, preferablyabout 0.5 to 1 grams of the spray-foam are applied.

As a vaginal formulation, the active ingredients can be used inconjunction with a spermicide, and may be employed with a condom,diaphragm, sponge or other contraceptive device. Examples of suitablespermicides include nonylphenoxypolyoxyethylene glycol (nonoxynol 9),benzethonium chloride, and chlorindanol. Suitably, the pH of thecomposition is 4.5 to 8.5. Vaginal compositions preferably have a pH of4.5 to 6, most preferably about 5.

Vaginal formulations also include suppositories (for example,gel-covered creams), tablets and films. The suppositories can beadministered by insertion with an applicator using methods well known inthe art.

The compositions can also be in the form of a time-release composition.In this embodiment, the composition is incorporated in a compositionwhich will release the active compound at a rate which will result inthe vaginal or anal concentration described above. Time-releasecompositions are disclosed in Controlled Release of Pesticides andPharmaceuticals, D. H. Lew, Ed., Plenum Press, New York, 1981; and U.S.Pat. Nos. 5,185,155; 5,248,700; 4,011,312; 3,887,699; 5,143,731;3,640,741; 4,895,724; 4,795,642; Bodmeier et al, Journal ofPharmaceutical Sciences, vol. 78 (1989); Amies, Journal of Pathology andBacteriology, vol. 77 (1959); and Pfister et al, Journal of ControlledRelease, vol. 3, pp. 229-233 (1986), all of which are incorporatedherein by reference.

The compositions can also be in the form which releases the compositionin response to some event such as vaginal or anal intercourse. Forexample, the composition may contain the vesicles or liposomes which aredisrupted by the mechanical action of intercourse. Compositionscomprising liposomes are described in U.S. Pat. No. 5,231,112 and Deamerand Uster, “Liposome Preparation: Methods and Mechanisms” in Liposomes,pp. 27-51 (1983); Sessa et al, J. Biol. Chem., vol. 245, pp. 3295-3300(1970); Journal of Pharmaceutics and Pharmacology, vol. 34, pp. 473-474(1982); and Topics in Pharmaceutical Sciences, D. D. Breimer and P.Speiser, Eds., Elsevier, New York, pp. 345-358 (1985), which areincorporated herein by reference.

The compositions can be associated with a contraceptive device orarticle, such as a vaginal ring device, an intrauterine device (IUD),vaginal diaphragm, vaginal sponge, pessary, condom, etc. In the case ofan IUD or diaphragm, time-release and/or mechanical-release compositionsmay be preferred, while in the case of condoms, mechanical-releasecompositions are preferred.

Rings and intravaginal sponges which release the therapeutic agents in atime-controlled fashion can be used. Suitable intravaginal sponges aredisclosed in U.S. Pat. Nos. 3,916,898 and 4,360,013, which areincorporated herein by reference. The present article may also be avaginal dispenser, such as those disclosed in U.S. Pat. No. 4,961,931,which is incorporated herein by reference.

The compositions can also be in the form of an intra-vaginal pill, anintra-rectal pill, or a suppository. The suppository or pill should beinserted into the vaginal or rectal cavity in a manner that permits thesuppository or pill, as it dissolves or erodes, to coat the vaginal orrectal walls with a prophylactic layer of the therapeutic agents.

In one particular embodiment, the composition contains nonoxynol, awidely-used spermicidal surfactant.

The compositions can also contain a lubricant that facilitatesapplication of the composition to the desired areas of skin andepithelial tissue, and reduces friction during sexual intercourse. Inthe case of a pill or suppository, the lubricant can be applied to theexterior of the dosage form to facilitate insertion.

Non-limiting examples of useful lubricants include cetyl esters wax,hydrogenated vegetable oil, magnesium stearate, methyl stearate, mineraloil, polyoxyethylene-polyoxypropylene copolymer, polyethylene glycol,polyvinyl alcohol, sodium lauryl sulfate, white wax, or mixtures of twoor more of the above. The amount of lubricant in the topical formulationcan range from about 0 to about 95 weight percent. Typical cream andointment formulations comprise 0.1 to 95 weight percent of lubricant.The topical formulations can comprise one or more adjuvants, wherein theadjuvant is an antimicrobial agent, antioxidant, humectant oremulsifier, or mixture of two or more thereof. The gels and foams of thepresent invention can include one or more antimicrobial agents andoptionally can include one or more of antioxidants, humectants andemulsifiers.

Non-limiting examples of useful antimicrobial agents are benzyl alcohol,propylene glycol, propyl paraben, methyl paraben, or mixtures of two ormore thereof. The amount of antimicrobial agents in the topicalformulation can range from about 0.01 to about 10 weight percent, and insome embodiments from about 0.2 to about 10 weight percent, on a basisof total weight of the topical formulation.

Non-limiting examples of useful antioxidants include butylatedhydroxyanisole, butylated hydroxytoluene, edetate disodium or mixturesof two or more thereof. The amount of antioxidant in the topicalformulation can range from about 0.01 to about 1 weight percent, and insome embodiments from about 0.01 to about 0.1 weight percent, on a basisof total weight of the topical formulation.

Non-limiting examples of useful humectants include ethylene glycol,glycerin, sorbitol or mixtures of two or more thereof. The amount ofhumectant in the topical formulation can range from about 1 to about 30weight percent, and in some embodiments from about 2 to about 20 weightpercent, on a basis of total weight of the topical formulation.

Non-limiting examples of useful emulsifiers include acrylic acidpolymers (such as carbomer brand thickeners e.g. Carbomer 934P,manufactured by Voveon, Inc.), polyoxyethylene-10-stearyl ether,polyoxyethylene-20-stearyl ether, cetostearyl alcohol, cetyl alcohol,cholesterol, diglycol stearate, glyceryl monostearate, glycerylstearate, polygeyceryl-3-oleate, hydroxypropyl cellulose,hydroxypropylmethyl cellulose, lanolin, polyoxyethylene lauryl ether,methyl cellulose, polyoxyethylene stearate, polysorbate, propyleneglycol monostearate, sorbitan esters, stearic acid or mixtures of two ormore thereof.

The amount of emulsifier in the topical formulation can range from about1 to about 40 weight percent, and in some embodiments from about 5 toabout 30 weight percent, on a basis of total weight of the topicalformulation.

The gel formulations of the present invention comprise one or moregelling agents. Non-limiting examples of useful gelling agents includecarboxylic acid polymers including acrylic acid polymers crosslinkedwith cross links such as allyl ethers of sucrose (e.g. carbomer brandthickeners), cetostearyl alcohol, hydroxymethyl cellulose,polyoxyethylene-polyoxypropylene copolymer, sodiumcarboxymethylcellulose, polyvinyl pyrrolidone, or mixtures of two ormore thereof. The amount of gelling agent in the topical gel formulationcan range from about 0.1 to about 10 weight percent, and in someembodiments from about 0.1 to about 1 weight percent, on a basis oftotal weight of the topical formulation.

The formulations can contain one or more additional excipients wellknown in the art, for example water and a thickening agent such ascolloidal silicon dioxide.

Ocular Formulations

Ocular formulations are typically in the form of eye drops, which mayinclude lubricants and thickeners, as well as pH buffered solutions.When used to treat eye disorders, the formulations can include, inaddition to the glutathione, organic acid and pharmaceuticallyacceptable salts, prodrugs and derivatives thereof, and sodiumbicarbonate, agents useful for treating ocular disorders.

For example, in treating ocular infections, such as trachoma,antimicrobials can be used. The antimicrobials can be antibacterials,antivirals, or antifungals, depending on the nature of the infection.The antimicrobials can treat the underlying disorder, and the othercomponents can restore and maintain homeostasis in the eye as it ishealing. Suitable antimicrobials are well known to those of skill in theart.

Eye infections and injuries are often associated with inflammation, soan anti-inflammatory, such as a steroid, for example, a corticosteroid,can also be used. By “inflammatory disease” is meant a disease statecharacterized by (1) alterations in vascular caliber that lead to anincrease in blood flow, (2) structural changes in the microvasculaturethat permit the plasma proteins and leukocytes to leave the circulation,and (3) emigration of the leukocytes from the microcirculation and theiraccumulation in the focus of injury. The classic signs of acuteinflammation are erythema, edema, tenderness (hyperalgesia), and pain.Chronic inflammatory diseases are characterized by infiltration withmononuclear cells (e.g., macrophages, lymphocytes, and plasma cells),tissue destruction, and fibrosis. Non-limiting examples of inflammatoryocular diseases include trachoma, wet and dry age-related maculardegeneration (AMD), diabetic retinopathy (DR), glaucoma, neovascularglaucoma, retinal vasculitis, uveitis, such as posterior uveitis,conjunctivitis, retinitis secondary to glaucoma, episcleritis,scleritis, optic neuritis, retrobulbar neuritis, ocular inflammationfollowing ocular surgery, ocular inflammation resulting from physicaleye trauma, cataract, ocular allergy and dry eye.

In some embodiments of this invention, wherein the composition isintended for topical administration to ocular or periocular tissues, thecomposition may be formulated for application as a liquid drop,ointment, a viscous solution or gel, a ribbon, or a solid. Thecomposition can be topically applied, for example, without limitation,to the front of the eye, under the upper eyelid, on the lower eyelid andin the cul-de-sac.

An exemplary composition for administration to the ocular and perioculartissues is an aqueous polymeric suspension. The polymeric suspendingagent is suspended in an aqueous medium. The polymeric suspending agentis preferably in suspension (i.e. water insoluble and/or waterswellable), although water soluble suspending agents are also suitablefor use with a suspension of the active agents. The suspending agentserves to provide stability to the suspension and to increase theresidence time of the dosage form on the eye. It can also enhance thesustained release of the drug in terms of both longer release times anda more uniform release curve.

Examples of polymeric suspending agents include dextrans, polyethyleneglycols, polyvinylpyrolidone, polysaccharide gels, Gelrite®, cellulosicpolymers like hydroxypropyl methylcellulose, and carboxy-containingpolymers such as polymers or copolymers of acrylic acid, as well asother polymeric demulcents. A preferred polymeric suspending agent is awater swellable, water insoluble polymer, especially a crosslinkedcarboxy-containing polymer.

Such polymers may be crosslinked by a polyfunctional crosslinking agent,preferably a difunctional crosslinking agent. The amount of crosslinkingshould be sufficient to form insoluble polymer particles, but not sogreat as to unduly interfere with sustained release of the activeagents. Typically the polymers are only lightly crosslinked. Preferablythe crosslinking agent is contained in an amount of from about 0.01% toabout 5%, preferably from about 0.1% to about 5.0%, and more preferablyfrom about 0.2% to about 1%, based on the total weight of monomerspresent. Included among such crosslinking agents are non-polyalkenylpolyether difunctional crosslinking monomers such as divinyl glycol;2,3-dihydroxyhexa-1,5-diene; 2,5-dimethyl-1,5-hexadiene; divinylbenzene;N,N-diallylacrylamide; N,N-diallymethacrylamide and the like. Alsoincluded are polyalkenyl polyether crosslinking agents containing two ormore alkenyl ether groupings per molecule, preferably alkenyl ethergroupings containing terminal H₂C═C< groups, prepared by etherifying apolyhydric alcohol containing at least four carbon atoms and at leastthree hydroxyl groups with an alkenyl halide such as allyl bromide orthe like, e.g., polyallyl sucrose, polyallyl pentaerythritol, or thelike; see, e.g., Brown U.S. Pat. No. 2,798,053, the entire contents ofwhich are incorporated herein by reference. Diolefinic non-hydrophilicmacromeric crosslinking agents having molecular weights of from about400 to about 8,000, such as insoluble di- and polyacrylates andmethacrylates of diols and polyols, diisocyanate-hydroxyalkyl acrylateor methacrylate reaction products of isocyanate terminated prepolymersderived from polyester diols, polyether diols or polysiloxane diols withhydroxyalkylmethacrylates, and the like, can also be used as thecrosslinking agents; see, e.g., Mueller et al. U.S. Pat. Nos. 4,192,827and 4,136,250, the entire contents of each patent being incorporatedherein by reference.

The crosslinked carboxy-vinyl polymers may be made from a carboxy-vinylmonomer or monomers as the sole monoethylenically unsaturated monomerpresent, together with a crosslinking agent or agents. Preferably thepolymers are ones in which up to about 40%, and preferably from about 0%to about 20% by weight, of the carboxy-containing monoethylenicallyunsaturated monomer or monomers has been replaced by one or morenon-carboxyl-containing monoethylenically unsaturated monomer ormonomers containing only physiologically and ophthalmically innocuoussubstituents, including acrylic and methacrylic acid esters such asmethyl methacrylate, ethyl acrylate, butyl acrylate,2-ethylhexylacrylate, octyl methacrylate, 2-hydroxyethyl-methacrylate,3-hydroxypropylacrylate, and the like, vinyl acetate,N-vinylpyrrolidone, and the like; see Mueller et al. U.S. Pat. No.4,548,990 for a more extensive listing of such additionalmonoethylenically unsaturated monomers.

Particularly preferred polymers are lightly crosslinked acrylic acidpolymers wherein the crosslinking monomer is 2,3-dihydroxyhexa-1,5-dieneor 2,3-dimethylhexa-1,5-diene. Preferred commercially available polymersinclude polycarbophil (Noveon AA-1) and Carbopol®. Most preferably, acarboxy-containing polymer system known by the tradename DuraSite®,containing polycarbophil, which is a sustained release topicalophthalmic delivery system that releases the drug at a controlled rate,is used in the aqueous polymeric suspension composition of the presentinvention.

The crosslinked carboxy-vinyl polymers used in practicing this inventionare preferably prepared by suspension or emulsion polymerizing themonomers, using conventional free radical polymerization catalysts, to adry particle size of not more than about 50 μm in equivalent sphericaldiameter; e.g., to provide dry polymer particles ranging in size fromabout 1 to about 30 μm, and preferably from about 3 to about 20 μm, inequivalent spherical diameter. Using polymer particles that wereobtained by mechanically milling larger polymer particles to this sizeis preferably avoided. In general, such polymers will have a molecularweight which has been variously reported as being from about 250,000 toabout 4,000,000, and from 3,000,000,000 to 4,000,000,000.

In a more preferred embodiment of the invention for topical ophthalmicadministration, the particles of crosslinked carboxy-vinyl polymer aremonodisperse, meaning that they have a particle size distribution suchthat at least 80% of the particles fall within a 10 μm band of majorparticle size distribution. More preferably, at least 90% and mostpreferably at least 95%, of the particles fall within a 10 μm band ofmajor particle size distribution. Also, a monodisperse particle sizemeans that there is no more than 20%, preferably no more than 10%, andmost preferably no more than 5% particles of a size below 1 μm. The useof a monodispersion of particles will give maximum viscosity and anincreased eye residence time of the ophthalmic medicament deliverysystem for a given particle size. Monodisperse particles having aparticle size of 30 μm and below are most preferred. Good particlepacking is aided by a narrow particle size distribution.

The aqueous polymeric suspension normally contains the active agents inan amount from about 0.05% to about 25%, preferably about 0.1% to about20%, more preferably about 0.5% to about 15%, more preferably about 1%to about 12%, more preferably about 2% to about 10.0%, and polymericsuspending agent in an amount from about 0.05% to about 10%, preferablyabout 0.1% to about 5% and more preferably from about 0.2% to about 1.0%polymeric suspending agent. In the case of the above described waterinsoluble, water-swellable crosslinked carboxy-vinyl polymer, anotherpreferred amount of the polymeric suspending agent is an amount fromabout 0.5% to about 2.0%, preferably from about 0.5% to about 1.2%, andin certain embodiments from about 0.6% to about 0.9%, based on theweight of the composition. Although referred to in the singular, itshould be understood that one or 25 more species of polymeric suspendingagent, such as the crosslinked carboxy-containing polymer, can be usedwith the total amount falling within the stated ranges. In one preferredembodiment, the composition contains about 0.6% to about 0.8% of apolycarbophil such as NOVEON AA-1.

When water soluble polymers are used as the suspending agent, such ashydroxypropyl methylcellulose, the viscosity will typically be about 10to about 400 centipoise, more typically about 10 to about 200centipoises or about 10 to about 25 centipoise.

Steroids are one of the most commonly used medications for decreasingocular inflammation. By inhibiting the breakdown of phospholipids intoarachidonic acid, these agents act early on the inflammatory pathway.The most common side effects of this class of medications are cataractformation and glaucoma. Drugs such as loteprednol etabonate (Lotemax;Bausch+Lomb, Rochester, N.Y.) carry a lower risk of increased IOP.1Another new agent is difluprednate (Durezol; Sirion Therapeutics, Tampa,Fla.), which possesses even greater potency than the other availablecorticosteroids.

Although nonsteroidal anti-inflammatory drugs have been used to treatinflammatory conditions, physicians should exercise caution whenprescribing this class of medications. In patients with severeinflammation combined with dry eye disease, treatment with non-steroidalanti-inflammatory drugs has caused corneal melting (Isawi and Dhaliwal,“Corneal melting and perforation in Stevens Johnson syndrome followingtopical bromfenac use,” J Cataract Refract Surg. 2007; 33(9):1644-1646).In contrast, cyclosporine 0.05% (Restasis; Allergan, Inc., Irvine,Calif.) has been shown to effectively control many causes of ocularsurface inflammation, and this ophthalmic emulsion has an excellentsafety profile. Accordingly, combinations of the active agents describedherein and cyclosporine, particularly in the form of ocular formulationssuch as eye drops, are also within the scope of the invention.

If additional therapy is required, autologous serum tears can be veryeffective. Because they contain several important components of naturaltears such as epidermal growth factor, fibronectin, and vitamin A,autologous serum tears increase the health of the ocular surface(Kojima, et al., “Autologous serum eye drops for the treatment of dryeye diseases” Cornea, 27(suppl 1):S25-30 (2008)).

Another alternative is to use agents such as tacrolimus, Sirolimus, andthe like, for example, in the form of a dermatologic ointment (Protopic;Astellas Pharma US, Inc., Deerfield, Ill.) (Wyrsch et al., “Safety oftreatment with tacrolimus ointment for anterior segment inflammatorydiseases,” Klin Monatsbl Augenheilkd, 226(4):234-236 (2009)). Thus,combinations of these agents and the glutathione, organic acid or saltsthereof, and sodium bicarbonate, are also within the scope of theinvention.

Methods of Treatment

The compositions described herein can be used to treat disordersassociated with mucosal membranes, by delivering the compositions to theappropriate mucosal membranes. In some embodiments, the mucosalmembranes are the lungs, such as the deep lung (alveolar region), and inother embodiments, the mucosal membranes are one or more of the eyes,mouth, nose, rectum, and vagina.

In still further embodiments, the composition further comprises, or thecomposition is administered in combination or in alternation with, oneor more additional therapeutic agents. That is, in some embodiments, thecomposition and further therapeutic agents are directed to the samelocus in the same formulation, and in other embodiments, the compositioncan be administered via one pathway, and the further therapeutic agentscan be administered via a different pathway.

In some embodiments, the further therapeutic agents treat the desireddisorder for which they are administered, but cause certain sideeffects, such as a drying of the mucosal membranes that results indiscomfort and/or injury, that can be addressed by administering thecompositions described herein.

In other embodiments, the further therapeutic agents and thecompositions described herein both treat the underlying disorder, thoughvia different means, such that an additive or synergistic effect can beachieved. As a result, in some aspects of this embodiment, lower dosesof the additional therapeutic agent can be effective, which lower dosescan result in fewer side effects, or provide other benefits to thepatient.

Representative further therapeutic agents include an antibiotic, anantifungal, a beta antagonist an immunosuppressant, an immunomodulatorand/or a steroid, in any combination.

Where the additional therapeutic agent is present, the resulting newformulations can provide new applications for the therapeutic agent,enhance the efficacy of the therapeutic agent, reduce unwanted sideeffects associated with the therapeutic agent, and/or reduce the dose ofthe therapeutic agent.

Particularly where the compositions include these therapeutic agents,the compositions can be used to treat mucosal diseases, pulmonarydiseases, autoimmune diseases (including multiple sclerosis, Crohn'sdisease, ulcerative colitis, lupus, inflammatory bowel syndrome,irritable bowel syndrome, etc.), infectious diseases (e.g., HIV),Alzheimer's disease, and the like.

In some aspects of these embodiments, a single formulation includes thetherapeutic agents and the glutathione, organic acid and bicarbonate,and in other aspects of these embodiments, the therapeutic agents arepresent in a first formulation and the glutathione, organic acid andbicarbonate are present in a second formulation. As such, thecompositions can be used in combination or ‘kit’ therapies. Theformulation with the therapeutic agent can be present in oral,injectable, or inhaled forms, and the glutathione, organic acid andbicarbonate present in an inhaled (e.g., pulmonary or intranasal)formulation.

The present invention finds use in both veterinary and medicalapplications. Suitable subjects of the present invention include, butare not limited to mammals. The term “mammal” as used herein includes,but is not limited to, primates (e.g., simians and humans), non-humanprimates (e.g., monkeys, baboons, chimpanzees, gorillas), bovines,ovines, caprines, ungulates, porcines, equines, felines, canines,lagomorphs, pinnipeds, rodents (e.g., rats, hamsters, and mice), etc. Insome embodiments of the present invention, the subject is a human. Humansubjects include both males and females and subjects of all agesincluding neonatal, infant, juvenile, adolescent, adult, and geriatricsubjects. In further embodiments of this invention, a subject can be aplant.

Yet further embodiments of this invention include the use of apharmaceutical composition of this invention in the manufacture of amedicament for treating a mucosal tissue disorder, treating an infectionin mucosal tissue and/or treating inflammation in mucosal tissue in asubject.

In particular embodiments, the pharmaceutical composition of thisinvention is administered one or more times daily. (e.g., 1, 2, 3, 4, 5,6, 7, 8, 9, 10 or more times a day)

In particular embodiments, the subject is a human.

In various embodiments of the methods of this invention, thepharmaceutical composition can be administered via inhalation,intranasally, via the eye, via the ear, via sinus irrigation, viabronchoscope or any combination thereof.

Treatment of Pulmonary Disorders

Thus, in one embodiment, the present invention provides a method oftreating an airway disorder or disease in a subject in need thereof,comprising delivering to the subject an effective amount of thepharmaceutical composition of this invention.

In the method described above, the airway disorder or disease can be,but is not limited to, chronic inflammatory lung disease, pulmonaryfibrosis, pulmonary vasculitis, pulmonary sarcoidosis, inflammationand/or infection associated with lung transplantation, acute lungrejection, pulmonary artery hypertension, bronchitis, sinusitis, asthma,cystic fibrosis, bacterial infection (e.g., by Pseudomonas aeruginosa,anthrax, Mycobacterium) fungal infection (e.g., by Aspergillus,Pneumocystis carnii), parasite infection, viral infection, chronicobstructive pulmonary disease (COPD), bronchiolitis obliterans syndrome(BOS), primary ciliary dyskinesia (PCD), idiopathic pulmonary fibrosis(IPF), alveolar protienosis, eosinophilic pneumonia, eosinophilicbronchitis, acute respiratory distress syndrome (ARDS), inflammationand/or infection associated with mechanical ventilation,ventilator-associated pneumonia, asbestos-related airway disorder ordisease, dust-related airway disorder or disease, silicosis, chemicalagent-related airway disease or disorder and any combination thereof.

As one nonlimiting example, the compositions and formulations of thisinvention can be administered to a subject in combination with aphosphodiesterase 3 (PDE3) inhibitor and/or a phosphodiesterase 4 (PDE4inhibitor. It is contemplated that the combination will allow for thePED3 and/or PED4 inhibitor to have a therapeutic effect in the presenceof the composition or formulation of this invention at a dose that islower than a dose that would have a similar therapeutic effect in theabsence of the composition or formulation of this invention.

Further embodiments of this invention include a method of treating aninfection in the airway of a subject (e.g., a subject in need thereof),comprising delivering to the subject an effective amount of thepharmaceutical composition of this invention.

The present invention also provides a method of treating inflammation inthe airway of a subject (e.g., a subject in need thereof), comprisingdelivering to the subject an effective amount of the pharmaceuticalcomposition of this invention.

While not wishing to be bound by a particular theory, it is believedthat the formulations disclosed herein are effective in achieving andmaintaining either a normal lung mucosa, or at least a more normal lungmucosa, which is an important factor in maintaining lung health. Drugsadministered to the lungs are often associated with certain sideeffects, in some cases because of dosage, and in other cases becausethey damage the lung tissue. In some embodiments, therapeutic agentscombined with the formulations disclosed herein are effective at lowerdoses, and at such lower doses, the incidence of side effects can bereduced. For example, one can decrease inhaled corticosteroid (ICS)dosing and, accordingly, reduce the risk of pneumonia in chronicobstructive pulmonary disease (COPD) patients, and reduce the dose ofβ-agonists and other bronchodilators to reduce the risk of death inasthma patients.

In other embodiments, where the therapeutic agent interacts unfavorablywith lung tissue, the formulations described herein can help to restorehomeostasis to the lung tissue, and thus help minimize or eliminatedamage caused by the therapeutic agents.

The present invention also provides a method of treating an airwaydisorder or disease in a subject in need thereof, comprising deliveringto the subject an effective amount of the pharmaceutical compositionsdescribed herein, optionally in combination with a steroid and/orbronchodilator, such as a beta₂-agonist.

Furthermore, the present invention provides a method of treating aninfection in the airway of a subject in need thereof, comprisingdelivering to the subject an effective amount of the pharmaceuticalcomposition described herein, optionally in combination with one or moreantibiotics. The antibiotics can be administered locally to the lungsand/or systemically

Inflammation in the airway of a subject in need thereof can be treatedby delivering to the subject an effective amount of the pharmaceuticalcompositions described herein.

In particular embodiments of the methods of this invention, uponadministration of the pharmaceutical composition to a human subject, theconcentration of thiocyanate in the airway surface liquid of the subjectis from about 0.5 mM to about 3.0 mM. The concentration of thiocyanatein the saliva of a normally healthy subject is from about 0.5 mM toabout 3.0 mM (Schultz et al, 1996); whereas, airway secretions ofnormally healthy individuals have been determined to contain 0.3 to 0.65mM SCN—(Wijkstrom-Frei et al., 2003). The nasal airway fluidconcentrations range from 0.1 to 1.2 mM. These values are more thantenfold greater than serum values (0.05 mM, Lundquist et al. 1995). Theamount to be delivered to the selected mucosal surface will be designedto result in concentrations up to 3.0 mM.

In particular embodiments of the methods of this invention, uponadministration of the pharmaceutical composition to a human subject, theconcentration of glutathione in the airway surface liquid of the subjectis from about 0.1 mM to about 1.0 mM. This is approximately 140 foldhigher than found in plasma in the same individual. The concentrationsdelivered are designed to raise the levels to 1.0 mM.

In some embodiments of the methods of this invention, the glutathione,pharmaceutically acceptable salt thereof, derivative thereof, analoguethereof and/or prodrug thereof, the organic acid, pharmaceuticallyacceptable salt thereof, derivative thereof, analogue thereof and/orprodrug thereof, the buffer and/or the thiocyanate of the pharmaceuticalcomposition can be administered or delivered as separate components butin a sequential and/or temporal manner that allows the components towork together to achieve the desired therapeutic effect. In a furtherembodiment the components can be delivered simultaneously from separatecontainers that feed into a common conduit that then delivers thecomponents to the subject.

In some embodiments, the pharmaceutical composition is delivered to theupper third of the nasal cavity, to the superior meatus, the olfactoryregion and/or the sinus region of the nose. The olfactory region is asmall area that is typically about 2-10 cm² in man (25 cm² in the cat)located in the upper third of the nasal cavity for deposition andabsorption by the olfactory epithelium and subsequent transport byolfactory receptor neurons. Located on the roof of the nasal cavity, inthe superior meatus, the olfactory region is useful for delivery in someembodiments, because it is the only known part of the body in which anextension of the CNS comes into contact with the environment (Bois etal. Fundamentals of Otolaryngology, p. 184, W. B. Saunders Co.,Philadelphia, 1989).

The compositions of the present invention are administered in a mannercompatible with the dosage formulation in such an amount as will beeffective for the desired result. In particular embodiments, thepharmaceutical composition is administered to the subject in atherapeutically effective amount (as described hereinabove). Thequantity to be administered depends on a number of factors, such as,e.g., the subject to be treated and the severity of the condition.Precise amounts of active ingredient required to be administered maydepend on the judgment of the practitioner. In general, the dose persubject may be 5 μg, 50 μg, or 250 μg, up to 5 mg, 10 mg, 20 mg, or 100mg, per dose.

Exemplary dosages include from about 0.001, 0.01 or 0.1 to about 1, 5,10 or 20 mg/dose, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more timesdaily, two to four times weekly, weekly, two to three times monthly ormonthly, or as needed by the subject.

The composition of this invention can be administered for a sustainedperiod, such as for at least about one month, at least about 2 months,at least about 3 months, at least about 6 months, or at least about 12months or longer (e.g., as a chronic life-long treatment).

Any suitable dosing schedule can be followed. For example, the dosingfrequency can be a once weekly dosing. The dosing frequency can be aonce daily or multiple times daily dosing. The dosing frequency can bemore than once weekly dosing. The dosing frequency can be more than oncedaily dosing, such as any one of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than10 daily doses. The dosing frequency can be intermittent (e.g., multipledaily dosing for 7 days followed by no doses for 7 days, repeated forany 14 day time period, such as 2 months, 4 months, 6 months or more).The dosing frequency can be continuous (e.g., one weekly dosing forcontinuous weeks).

In some embodiments, the pharmaceutical composition of this inventioncan be administered in a single “shock” dose, for example, during abronchoscopy.

In other embodiments, the methods of the invention can be carried out onan as-needed basis by self-medication.

Any of the dosing frequencies can be used with any dosage amount.Further, any of the dosing frequencies and/or dosage amounts can be usedwith any of the pharmaceutical compositions described herein.

The pharmaceutical composition can be delivered in any suitable volumeof administration, In representative embodiments of the invention, theadministration volume for intranasal delivery ranges from about 25microliters to 200 microliters or from about 50 to 150 microliters orfrom about 50, 100, 250 or 500 microliters to about 1, 2, 3, 3.5 or 4milliliters in a human. Typically, the administration volume is selectedto be large enough to allow for delivery of therapeutic quantities whileaccounting for dilution in ASL in maintenance conditions in relatively“normal” airways (10-30 ml ASL) and in cystic fibrosis (CF) airways(40-50 ml ASL or more plus thick, tenacious, and heavily infected mucussecretions).

“Administration by inhalation” or “delivery by inhalation” meansadministration to or delivery to the subject through the mouth and/ornose.

The term “intranasal administration” as used herein, refers to asystemic form of administration of a pharmaceutical composition of thisinvention, whereby the composition is introduced into one or both of thenasal passages of a subject such that the composition contacts the nasalmucosa and in some embodiments, is absorbed into the systemiccirculation. In certain embodiments, a therapeutically effective amountis administered. Intranasal administration of the pharmaceuticalcompositions of the present invention can comprise a singleadministration or multiple administrations of the compositions.

Intranasal administration of the pharmaceutical compositions of thepresent invention can be achieved by any known method. In particularembodiments, intranasal administration is by inhalation (e.g., using aninhaler, atomizer or nebulizer device), alternatively, by spray, tube,catheter, syringe, dropper, packtail, pipette, pledget, and the like. Asa further illustration, the pharmaceutical composition can beadministered intranasally as (1) nose drops, (2) powder or liquid spraysor aerosols, (3) liquids or semisolids by syringe, (4) liquids orsemisolids by swab, pledget or other similar means of application, (5) agel, cream or ointment, (6) an infusion, or (7) by injection, or by anymeans now known or later developed in the art. In particularembodiments, the method of delivery is by nasal drops, spray or aerosol.As used herein, aerosols can be used to deliver powders, liquids ordispersions (solids in liquid).

In representative embodiments, the pharmaceutical formulation isdirected upward during administration, so as to enhance delivery to theupper third (e.g., the olfactory epithelium in the olfactory region) andthe side walls (e.g., nasal epithelium) of the nasal cavity. Further,orienting the subject's head in a tipped-back position or orienting thesubject's body in Mygind's position or the praying-to-Mecca position canbe used to facilitate delivery to the olfactory region.

The formulations can be provided in single or multidose form. In thelatter case a means of dose metering can be provided. In the case of adropper or pipette this may be achieved by the patient or caregiveradministering an appropriate, predetermined volume of the composition.In the case of a spray this may be achieved, for example, by means of ametering atomizing spray pump.

A further aspect of the present invention is an intranasal spray devicecomprising a pharmaceutical composition of the present invention.

Many devices are known in the art for nasal delivery. Exemplary devicesinclude particle dispersion devices, bidirectional devices, and devicesthat use chip-based ink jet technologies. ViaNase (Kurve Technolgies,Inc., USA) uses controlled particle dispersion technology. (e.g., anintegrated nebulizer and particle dispersion chamber apparatus, forexample, as described in International patent publication WO2005/023335). Optinose and Optimist (OptiNose, AS, Norway) andDirectHaler (Direct-Haler A/S, Denmark) are examples of bidirectionalnasal delivery devices. Ink jet dispensers are described in U.S. Pat.No. 6,325,475 (MicroFab Technologies, Inc., USA) and use microdrops ofdrugs on a millimeter sized chip. Devices that rely oniontophoresis/phonophoresis/electrotransport are also known, asdescribed in U.S. Pat. No. 6,410,046 (Intrabrain International NV,Curacao, AN). These devices comprise an electrode with an attached drugreservoir that is inserted into the nose. Iontophoresis,electrotransport or phonophoresis with or without chemical permeationenhancers can be used to deliver the drug to the target region (e.g.,olfactory). Other commercially available nasal applicators are, forexample, the Pfeiffer unit dose and bidose system, the Valois monospray,bidose and monopowder system or the Becton-Dickinson Accuspray™ system.Also suitable are glass or plastic bottles with commercially availablemetering pump spray heads.

Nasal delivery devices are also described in U.S. Pat. No. 6,715,485(OptiNose AS); U.S. Pat. No. 6,325,475 (Microfab Technologies, Inc.);U.S. Pat. No. 6,948,492 (University of Kentucky Research Foundation);U.S. Pat. No. 6,244,573 (LyteSyde, LLC); U.S. Pat. No. 6,234,459(LyteSyde, LLC); U.S. Pat. No. 6,244,573 (LyteSyde, LLC); U.S. Pat. No.6,113,078 (LyteSyde, LLC); U.S. Pat. No. 6,669,176 (LyteSyde, LLC); U.S.Pat. No. 5,724,965 (Respironics Inc.); and U.S. Patent PublicationsUS2004/0112378 A1; US 2004/0112379 A1; US 2004/0149289 A1; US2004/0112380 A1; US 2004; 0182388 A1; US 2005/0028812 A1; US2005/0235992 A1; US 2005/0072430 A1 and US 2005/0061324 A1.

Further, the pharmaceutical compositions of the present invention canoptionally be administered in combination with one or more othertherapeutic agents, for example, other therapeutic agents useful in thetreatment and/or prevention of and/or any other treatment that may bebeneficial to the subject.

As used herein, the administration of two or more compounds “incombination” means that the two compounds are administered closelyenough in time that the presence of one alters the biological effects ofthe other. The two compounds may be administered concurrently, in thesame or different formulations, or sequentially. Concurrentadministration can be carried out by mixing the compounds prior toadministration, or by administering the compounds in two differentformulations, for example, at the same point in time but at differentanatomic sites or using different routes of administration. As usedherein, “concurrent” or “concurrently” means sufficiently close in timeto produce a combined effect (that is, concurrently can besimultaneously, or it can be two or more events occurring within a shorttime period before or after each other).

The present invention also provides a method of treating a disease ordisorder in mucosal tissue in a subject. Nonlimiting examples of mucosaltissue of this invention include the mouth, nose, eye, ear, upperrespiratory tract, lower respiratory tract, gastrointestinal tract,vagina, rectum and urethra. Nonlimiting examples of diseases and/ordisorders that can be treated according to the methods of this inventioninclude oral apthae, Behcet's Disease, mucosal discoid lupus (e.g.,Erythematosus), Bullous Diseases in mucosa (e.g., drug eruptions;Stevens-Johnsons Syndrome, Bullous lupus), herpes simplex and otherviral infections, thrush and other fungal infections, lichenoidmucositis (e.g., lichen planus), inflammatory bowel disorder, andcancer.

Treatment of Ocular Disorders

Oxidative damage, UV damage, dystrophies and degenerations (e.g.,macular degeneration) have been associated with decreased glutathioneand ascorbate. Accordingly, the compositions described herein canprovide lens, retina and corneal protection, repair and reducedinflammation, which can reduce the severity or incidence of diseasessuch as cataracts, dry eye and other retinopathies and ocular disordersthat are either primary or secondary to other disorders or drugreactions.

Some of these disorders have an inflammatory component, such astrachoma, wet and dry age-related macular degeneration (AMD), diabeticretinopathy (DR), glaucoma, neovascular glaucoma, retinal vasculitis,uveitis, such as posterior uveitis, conjunctivitis, retinitis secondaryto glaucoma, episcleritis, scleritis, optic neuritis, retrobulbarneuritis, ocular inflammation following ocular surgery, ocularinflammation resulting from physical eye trauma, cataract, ocularallergy and dry eye. When treating these disorders, the compositions canoptionally be administered with an anti-inflammatory agent.

The compositions described herein can be used, optionally but preferablyin combination with antimicrobial therapy, to treat or prevent a varietyof conditions associated with ocular infection. For example, conditionsof the eyelids, including blepharitis, blepharconjunctivies,meibomianitis, acute or chronic hordeolum, chalazion, dacryocystitis,dacryoadenities; conditions of the conjunctiva, includingconjunctivitis, ophthalmia neonatorum, and trachoma; conditions of thecornea, including corneal ulcers, superficial and interstitialkeratitis, keratoconjunctivitis, foreign bodies, and post operativeinfections; and conditions of the anterior chamber and uvea, includingendophthalmitis, infectious uveitis, and post operative infections, area few of the tissues and conditions that can be treated by topicalapplication of the therapeutic agents.

The prevention of infection includes pre-operative treatment prior tosurgery as well as other suspected infectious conditions or contact.Examples of prophylaxis situations include treatment prior to surgicalprocedures such as blepharoplasty, removal of chalazia, tarsorrhapy,procedures for the canualiculi and lacrimal drainage system and otheroperative procedures involving the lids and lacrimal apparatus;conjunctival surgery including removal of ptyregia, pingueculae andtumors, conjunctival transplantation, traumatic lesions such as cuts,burns and abrasions, and conjunctival flaps; corneal surgery includingremoval of foreign bodies, keratotomy, and corneal transplants;refractive surgery including photorefractive procedures; glaucomasurgery including filtering blebs; paracentesis of the anterior chamber;iridectomy; cataract surgery; retinal surgery; and procedures involvingthe extra-ocular muscles. The prevention of ophthalmia neonatorum isalso included.

Brain

Studies have indicated oxidative stress in the central nervous system(CNS) and brain contribute to diseases such as Alzheimer's, Parkinson's,ischemia and traumatic injuries, etc., to the brain and CNS. As such,this therapy could offer neuronal, glial and astrocyte protection,repair and reduced inflammation, which could reduce the severity orincidence of these diseases. In these embodiments, other therapeuticagents useful for treating these disorders, including dopamine agonistsand partial agonists, selective serotonin reuptake inhibitors (SSRI) canalso be used. In some aspects of these embodiments, the compositions areadministered intrathecally, and in other aspects, the compositions areadministered in another manner and cross the blood-brain barrier.

Nose

As an extension of the pulmonary and oral systems, application to thenasal passages can reduce nasal inflammation and infection, sinusinflammation and infection and other disorders primary or secondary toother disorders or drug reactions. Nasal and sinus administration ofthis therapy can lead to decreased pulmonary disease, as inflammation inthe sinuses (e.g., asthma and cystic fibrosis) is often linked toinflammation and infection in the lower airways.

Mouth

Glutathione and ascorbate play a critical role in innate immune factorsin the oral mucosa. This therapy can therefore offer oral, periodontaland dental protection, repair and reduced inflammation, which couldreduce the severity or incidence of oral diseases that are primarydisorders or secondary to other disorders or drug reactions.

In some embodiments, periodontal diseases can be treated withantibiotics, such as doxycycline and oral rinses such as chlorhexidine,which can be combined with the compositions described herein. As such,the therapy can treat the underlying disorder, and minimize/repairdamage to the oral mucosa.

When patients are suffering from viral disorders such as cold sores,shingles, aphthous ulcers, and the like, the compositions can alsoinclude, or be co-administered with, antiviral agents. Particularly whenthe patients suffer from pain in their mouth, whether from bacterial orviral causes, physical injuries, or oral surgery, includingtonsillectomies, uvulaplasties, scaling and root planning, grafting, andthe like, the formulations described herein can help maintainhomeostasis in the oral mucosa, which can accelerate healing, and caninclude anesthetics such as lidocaine, marcaine, xylocaine, and thelike, to help alleviate pain. Anti-inflammatory agents can also bepresent.

Ears

Glutathione and ascorbate play a critical role in innate immune factorsin the auditory system. This therapy can therefore offer auditoryprotection, repair and reduced inflammation, which could reduce theseverity or incidence of auditory diseases that are primary disorders(e.g., otitis media) or secondary to other disorders or drug reactions.

Upper GI Tract

Glutathione and ascorbate play a critical role in innate immune factorsin the upper GI tract. This therapy can therefore offer esophageal,gastric, hepatic, pancreatic, small intestinal, colonic and rectalprotection, repair and reduced inflammation, which could reduce theseverity or incidence of GI diseases that are primary disorders (e.g.,esophagitis, hepatitis C, pancreatitis, acid reflux) or secondary toother disorders (e.g., cystic fibrosis) or drug reactions.

Lower GI Tract

Glutathione and ascorbate play a critical role in innate immune factorsin the lower GI tract. This therapy can therefore offer colonic andrectal protection, repair and reduced inflammation, which could reducethe severity or incidence of GI diseases that are primary disorders(e.g., Crohn's, ulcerative colitis, inflammatory bowel disease) orsecondary to other disorders (e.g., cystic fibrosis) or drug reactions.

Urinary Tract

Glutathione and ascorbate play a critical role in innate immune factorsin the urinary tract. This therapy can therefore offer urinary tractprotection, repair and reduced inflammation, which could reduce theseverity or incidence of urinary tract diseases that are primarydisorders (e.g., bladder cancer, cystitis, urinary tract infections) orsecondary to other disorders (e.g., smoking or infection) or drugreactions.

Renal

Glutathione and ascorbate play a critical role in innate immune factorsin the renal system. This therapy can therefore offer renal protection,repair and reduced inflammation, which could reduce the severity orincidence of renal diseases that are primary disorders (e.g., kidneycancer) or secondary to other disorders (e.g., cystic fibrosis, drugtoxicity) or drug reactions. This therapy has the potential to reducedosages of other drugs thereby limiting exposure of the kidneys to toxicdrug levels.

Urogenital Tract

Glutathione and ascorbate play a critical role in innate immune factorsin the urogenital system. This therapy can therefore offer urogenitalprotection, repair and reduced inflammation, which could reduce theseverity or incidence of urogenital diseases that are primary disorders(e.g., HPV) or secondary to other disorders (e.g., menopause, Chlamydiainfection) or drug reactions (e.g., fungal infection). This therapy hasthe potential to reduce dosages of other drugs thereby limiting exposureof the urogenital tract to toxic drug levels.

Skin, Hands and Feet

Glutathione and ascorbate play a critical role in innate immune factorsin the skin, hands, finger and toe nails, and feet. This therapy couldoffer protection, repair and reduced inflammation to the skin and feet,which could reduce the severity or incidence of dermatological diseasesthat are primary disorders (e.g., bullous diseases, connective tissuediseases, i.e., lupus, allergic reaction, acne, eczema, psoriasis) orsecondary to other disorders (e.g., viral infection, UV damage) oradverse drug reactions (e.g., dry skin, mucositis, fungal infection).This therapy has the potential to reduce dosages of other drugs therebylimiting exposure of the skin and feet to noxious drug levels.

Circulatory System

Glutathione and ascorbate play a critical role in innate immune factorsin the circulatory system. This therapy can therefore offer protection,repair and reduced inflammation to the circulatory system, which couldreduce the severity or incidence of circulatory diseases that areprimary disorders (e.g., atherosclerosis) or secondary to otherdisorders (e.g., pulmonary hypertension, endocarditis) or drug reactions(e.g., azithromycin). This therapy has the potential to reduce dosagesof other drugs thereby limiting exposure of the circulatory system totoxic drug levels. For example, treating earlier stage chronicobstructive pulmonary disease (COPD) with inhaled corticosteroid therapycombined with a long-acting beta agonist, reduced cardiac deaths in posthoc data queries. Therefore decreasing inflammation in the discretesystem (e.g., lungs) may have systemic effects (e.g., circulatorysystem). The compositions and formulations of the present invention canbe used to establish and/or maintain homeostasis in the mucosalenvironment of the lungs of a smoker or former smoker to reduce the riskor incidence of lung cancer and/or smoking-related diseases ordisorders. The compositions and formulations of this invention can alsobe used in combination with therapeutic agents to treat lung cancerand/or smoking-related diseases or disorders, with the benefit that thetherapeutic agent is effective at a lower dose when used in combinationwith a composition or formulation of this invention.

Cancer

Cancer is caused by the dysregulation of multiple pathways and is oftenexacerbated by oxidative stress and the deactivation of innate immunedefenses. This therapy could offer protection, repair and reducedinflammation, which could reduce the severity or incidence of primarycancers (e.g., bladder, lung, breast) or cancers that are secondary toother disorders (e.g., thyroid, cervical, tonsilar) or drug reactions(e.g., immunosuppression, post-transplant lymphoma). This therapy hasthe potential to reduce dosages of other non-cancer related drugs,thereby limiting exposure to toxic drug levels. Additionally, it can beused in conjunction with cancer therapies to increase efficacy and/ordecrease side effects.

Anticancer therapy is known to be associated with dry mouth, soadministration of the compositions described herein to patientsundergoing chemotherapy can minimize damage to the oral mucosa caused bydry mouth.

Autoimmune Diseases

Autoimmune diseases are caused by dysregulation of the immune system andis often exacerbated by oxidative stress and the deactivation of innateimmune defenses. This therapy could offer protection, repair and reducedinflammation, which could reduce the severity or incidence of primaryautoimmune diseases (e.g., lupus, arthritis, Crohn's disease, ulcerativecolitis, multiple sclerosis, Type 1 diabetes) or autoimmune diseasesthat are secondary to other disorders (e.g., graft vs. host disease) ordrug reactions (e.g., chemotherapy, immunosuppression). This therapy hasthe potential to reduce dosages of other autoimmune disease-relateddrugs, thereby limiting exposure to toxic drug levels. Additionally, itcan be used in conjunction with other therapies to increase efficacyand/or decrease side effects.

Alzheimer's Disease

Alzheimer's disease is caused by genetic predisposition,neuroinflammation, disrupted neuronal communication, oxidative stressand possibly infectious agents. This therapy could offer protection,repair and reduced inflammation, which could reduce the severity orincidence of Alzheimer's disease or dementias that are secondary toother disorders (e.g., aging) or drug reactions (e.g., anesthesia,immunosuppression, chemotherapy). This therapy has the potential toreduce dosages of other Alzheimer's disease-related drugs, therebylimiting exposure to toxic drug levels. Additionally, it can be used inconjunction with other therapies to increase efficacy and/or decreaseside effects.

Fungal Infections

Fungal infections are considered opportunistic infections especially inimmunocompromised, cancer patients, and in diseases associated withsteroid application (e.g., inhaled corticosteroids). They can be bothcommunity- and hospital-acquired and are most commonly found to besecondary to immunosuppressive, antibiotic, or steroid therapy. Thistherapy supplies innate immune factors, the components needed to preventor treat primary fungal infections (e.g., athlete's foot) and reduce theincidence of or treat fungal infections secondary to other therapies(e.g., thrush). This therapy has the potential to reduce dosages ofother fungal infection-related drugs, thereby limiting exposure to toxicdrug levels. Additionally, it can be used in conjunction with othertherapies to increase efficacy and/or decrease side effects.

Viral Infections

Viral infections are common in the general population thoughimmunocompromised and cancer patients are most susceptible. This therapysupplies innate immune factors, the components needed to prevent ortreat primary viral infections (e.g., HIV, HPV, HSV, influenza) andreduce the incidence of or treat viral infections secondary to othertherapies (e.g., immunosuppression). This therapy has the potential toreduce dosages of other viral infection-related drugs, thereby limitingexposure to toxic drug levels. Additionally, it can be used inconjunction with other therapies to increase efficacy and/or decreaseside effects and prophylactically in the general population during fluseason. The methods and compositions of this invention can also beadministered to an immunocompromised subject and/or a subject in acommunity or hospital setting that renders the subject at increased riskof infection (e.g., an intubated patient, a nursing home resident, adialysis patient, etc.).

Bacterial Infections

Bacterial infections are common in the general population thoughimmunocompromised and cancer patients are most susceptible. They can beboth community- and hospital-acquired. This therapy supplies innateimmune factors, the components needed to prevent or treat primarybacterial infections (e.g., ventilator and other hospital and communityassociated pneumonias, otitis media, sinusitis) and reduce the incidenceof or treat bacterial infections secondary to other therapies (e.g.,immunosuppression). This therapy has the potential to reduce dosages ofother bacterial infection-related drugs, thereby limiting exposure totoxic drug levels. Additionally, it can be used in conjunction withother therapies to increase efficacy and/or decrease side effects.

Prophylaxis

In addition to providing methods of treatment, the compositionsdescribed herein can also be used to provide prevention of variousdiseases and disorders.

The formulations described herein can optionally be enhanced bysupplying methionine, cysteine and/or N-acetylcysteine and otherprecursors required for the intracellular production of glutathione.Ensuring adequate intracellular concentrations of glutathione by oral,intravascular or inhalation delivery of molecules required for itsintracellular production can:

1) ameliorate the effects of oxidant stress on the epithelium in theinflamed airway and

2) provide a supply of glutathione to be delivered to the mucosalsurface from the host's airway epithelium as the diseased epithelium, inthe non-cystic fibrosis condition, recovers.

Since cystic fibrosis patients cannot excrete glutathione from theirairway epithelium, an approach of intracellularly generating glutathionemay not be as effective for such patients as it would be for otherpatients.

While not wishing to be bound by a particular theory, it is believedthat there is a general depletion of glutathione and ascorbate, whichcauses inflammatory cells in chronic inflammatory conditions to generatelarge amounts of reactive nitrogen and oxygen species that damage hosttissues. Therefore, replenishing glutathione and ascorbate and theprecursors needed to generate them intracellularly would be predicted toameliorate the intensity of this inappropriate response by reducing thegeneration of these reactive species. The systemic delivery ofglutathione, ascorbate and their precursors can, therefore, modify theintensity of pathologic inflammation systemically. This approach cantherefore be used therapeutically and prophylactically in cysticfibrosis as well as other chronic conditions.

In the pulmonary area, for example, there are a number of smokingrelated disorders, such as COPD, cardiac disorders, urinary tractdisorders including cancer, gastrointestinal disorders such as pepticulcer disease, and the like, which can be prevented, at least to someextent, by administering the formulations prophylactically. Prophylacticadministration can establish and/or maintain homeostasis in the mucosaltissue, and help prevent damage to the tissue.

The compositions and formulations of this invention can also beadministered to a subject to ameliorate unwanted effects of othertherapies. For example, the compositions and formulations of thisinvention can be administered to a subject that is taking inhaledsteroids (ICS), which are linked to increased pneumonia risk in subjectswith chronic obstructive pulmonary disease (COPD), as well as anincreased risk of developing thrush, and/or to a subject that is takinga long acting beta antagonist (LABA), which is linked to increased riskof death in subjects with asthma. By administering the compositions andformulations of this invention to such subjects and thus establishing ormaintaining mucosal membranes in the subject in homeostasis, theeffective dose of the ICS or LABA can be reduced, thereby reducing thesubject's risk of these undesirable side effects.

The compositions can also be administered prophylactically during coldand flu season, so as to prevent viral infections in the lungs and othermucosal membranes. This is particularly true of medical workers, who areexposed to a variety of infectious agents, including viruses, bacteria,fungi, and the like. Where the mucosal membrane is maintained inhomeostasis, it can help fight off infectious diseases, so prophylacticadministration is particularly useful for such workers.

Subjects in areas with air pollution and/or contaminated air can beprotected against the effects of air pollution and/or contaminated airon the lungs and other mucosal membranes by prophylacticallyadministering the formulations to the desired mucosal membranes. Forexample, military personnel, fireman, factory workers, and the like thatlive and/or work in an air-polluted and/or contaminated environment(e.g., contaminated by gas, toxic vapors, chemical spill, smoke, dust,etc.) can benefit from prophylactic administration of the formulations.

Prophylactic administration of the formulations can also be useful whena subject is exposed to dry air and other airway-irritating and/orcontaminated environments, such as those experienced when traveling(e.g., on a plane, train, bus, car, etc.).

Prophylactic oral administration of the compositions and formulations ofthis invention can minimize dry mouth, regardless of its causes. Thecompositions for prophylaxis and/or treatment of dry mouth includetoothpastes, mouthwashes, gels, and creams. In addition to the activeingredients described herein, therapeutic agents, such as sodiummonofluorophosphate, enzymes such as glucose oxidase, lactoferrin,lactoperoxidase and lysozyme, and, to protect against the formation ofPBF (plaque biofilm), enzymes such as mutanase and dextranase can beadded.

The compositions and formulations of this invention can also beadministered to a subject undergoing radiation therapy to ameliorateradiation-induced mucositis, as well as to treat and/or preventinfection secondary to mucositis.

With respect to gastrointestinal (“GI”) administration, the formulationscan prevent certain GI disorders, including peptic ulcer disease, bymaintaining homeostasis in the gastrointestinal tract.

IPF exacerbations are linked to GI reflux and GERD (Acute Exacerbationsin Patients With Idiopathic Pulmonary Fibrosis, Respiratory Research,2013 Jun. 21), which can effect lung transplants. The compositionsdescribed herein can be administered to protect the airway from refluxdamage to the epithelium.

With respect to rectal or vaginal administration, maintaining themucosal membranes in homeostasis can prevent tears or other tissuedamage, which can lower the likelihood of contracting diseases such asHIV, herpes, syphilis, and the like.

With respect to organ transplants, one can minimize and/or preventdamage to certain tissues by contacting them with the compositions andformulations described herein. Accordingly, the components can be addedto perfusion liquids or used as components of a liquid used to slow thedeterioration of an organ intended for transplantation.

The present invention is explained in greater detail in the followingnon-limiting examples.

EXAMPLES Example 1 Preparation of Formulation 1

Glutathione (150 grams), ascorbic acid (10 grams) and sodium bicarbonate(45.7 grams) are added to a IL volumetric flask and brought to a finalvolume using deionized distilled water.

Example 2 Preparation of Formulation 2

Glutathione (150 grams), ascorbic acid (48.4 grams) and sodiumbicarbonate (84 grams) are added to a 1 L volumetric flask and broughtto a final volume using deionized distilled water.

Example 3 Treatment of a Patient Diagnosed with Asthma

Patient A was a 50 year old Caucasian female with severe asthmadiagnosed in childhood and who received continuous systemic steroids tomanage her condition since age 13. The patient underwent hospitalizationfor asthmatic exacerbations several times per year and reported dyspneaon exertion (DOE), greatly limiting her exercise tolerance even betweenexacerbations. After initiating inhalation therapy with Formulation 2,the patient expelled copious mucus plugs, and her forced expiratoryvolume in one second (FEV1) increased by 30%. After taking thismedication for 2 months, the patient reported that she felt betteroverall and is now able to run for an hour at a time without shortnessof breath.

Typical treatment entailed use of a nebulizer once or twice a day. Thestandard dose of Formulation 2 was a 4 ml volume nebulized for a tenminute period.

Example 4 Treatment of a Patient Diagnosed with Cystic Fibrosis

Patient B was a 41 year old Caucasian male diagnosed with cysticfibrosis (CF), severe lung disease and a clotting disorder. He did nottolerate and refused to use hypertonic saline and other standard inhaledtherapies because he felt they were either ineffective or, in the caseof hypertonic saline, induced a harsh, unproductive cough that oftenresulted in massive hemoptysis. This individual was significantly shortof breath with excursion prior to using the formulation. The patientinhaled Formulation 1 twice a day. Within a week of starting treatmentwith Formulation 1, the patient reported a significant decrease in DOEand reported being able to climb two flights of stairs withoutdifficulty, an activity he would not have considered attempting prior tostarting this therapy. This patient took Formulation 1 for approximately2 years (2007-2009) until he rapidly deteriorated and died secondary toan acute episode of massive hemoptysis associated with his coagulopathy.

The standard dose of Formulation 1 was a 4 ml volume nebulized for a tenminute period.

Example 5 Treatment of a Patient Diagnosed with Cystic Fibrosis

Patient C was a 51 year old Caucasian female diagnosed with cysticfibrosis. She began taking Formulation 1 by inhalation in 2007 afterrepeated extended hospitalizations for CF exacerbations requiringintravenous antibiotics associated with deteriorating lung function.Since beginning this therapy, the patient has only occasionally requiredhospitalizations for 3-4 days. Once during the five year period, thepatient reported that this inhaled formulation “stopped working.”Investigation revealed that the patient mistakenly received 25% of theprescribed dose during this period. Reinstitution of the full dosealleviated her symptoms. Additionally, this patient reported that afterabout two weeks, even the correct formulation began to loseeffectiveness. Formulation 2 is now freshly prepared every two weeks forPatient C. There has been no perceived clinical deterioration ofFormulation 2 when it is freshly prepared every two weeks.

Example 6 Treatment of a Patient Diagnosed with Bronchiolitis ObliteransSyndrome

Patient D was a 28 year old Caucasian female diagnosed with end-stage CFlung disease. The patient had undergone lung transplant evaluation atanother hospital and was admitted to our hospital for severe bilateralpneumonia. The patient was taking nebulized albuterol, dornase alfa,hypertonic saline, and tobramycin (TOBI). She routinely experiencedhemoptysis using these and other nebulized antibiotics. She found theinhalation of hypertonic saline especially difficult to tolerate becauseit often induced a violent non-productive cough and vomiting. In anattempt to address the patient's suffocating, copious, and viscousairway secretions, she began treatment with Formulation 1 in November2010. Inhalation treatment was carried out 2-6 times a day until sheunderwent a bilateral lung transplant in March 2011. The patientreported that this treatment was the only “source of relief,” Thepatient reported having administered it whenever “I felt too congestedto walk around or function.” She felt this formulation was very welltolerated and that it stabilized her condition.

The same patient, after her lung transplant, began taking the inhaledformulation one year post transplant in an attempt to address recurrentepisodes of infection and A1 rejection characterized by T-cellinfiltration on transbronchial biopsy and persistent severe airwayinflammation characterized by an erythematous, friable mucosa, copiousmucopurulent secretions, and airway mucus plugging. The recurrent acuteepisodes were treated with methylprednisolone, prednisone tapers, rabbitanti-thymocyte globulin, and multiple courses of oral and IVantibiotics. Although these episodes of rejection resolved, forced vitalcapacity (FVC) never exceeded 95% of expected values, FEV1 neverexceeded 85% of predicted, and mid-volume flow rates (FEF25-75%) neverexceeded 40% of predicted. Chest computerized tomography (CT) scansrevealed persistent ground-glass opacity bilaterally in the lower lobes.Several months after receiving the rabbit anti-thymocyte globulin, thepatient reported left-sided chest tightening. Bronchoscopy demonstratedA1 rejection, Aspergillus infection, Pseudomonas infection, decreasedsmall airway function, friable airway tissue, and copious purulentsecretions with mucus plugging. After treatment with methylprednisolone,prednisone taper, antifungals, and antibiotics, lung function recoveredto levels reported above but a chest CT again demonstrated bilaterallower lobe ground-glass opacities. Bronchoscopy again revealed aninflamed erythematous friable mucosa with copious mucopurulent airwaysecretions and complete obstruction of the left lower lobe by viscousmucus. Upon first treatment with the inhaled Formulation, the patientexpectorated mucus plugs. Two months after beginning this therapybronchoscopy revealed a normal-appearing airway with resolution of allthe pathology previously noted and a chest CT demonstrated resolution ofthe previously noted ground-glass opacities. Two months after theinitial dose, the patient's pulmonary function tests had improved andwere essentially normal with a FVC of 101% of predicted, FEV1 93% ofpredicted, and FEF25-75% of 73% of predicted. Clinically, the patientreported resolution of the previously noted chest tightness and hasnoted increased exercise tolerance.

Cystic fibrosis transmembrane conductance regulator (CFTR) playscritical roles in the regulation of inflammation and infection of theairway. The absence of functioning CFTR is either directly or indirectlyassociated with deficiencies in several specific anions in the exocrinesecretions that constitute the airway surface liquid. These anions,including glutathione, ascorbate, bicarbonate and thiocyanate, areessential cofactors for several innate systems essential to themaintenance of a homeostatic balance on mucosal surfaces. It is theintent, with some embodiments of this invention, to restore the ioniccomposition in these airways to affect down-regulation of pathologicinflammatory responses, suppress neutrophil recruitment, redirect hostdestructive ROS and RNI to species with more selective antimicrobialactivity, alter the growth supportive environment for airway pathogens,and provide critical cofactors for the function of innate defensefactors including lactoperoxidase and lactoferrin. In some embodiments,this will be accomplished by inhaled delivery of nebulized liquidcontaining a combination of glutathione, bicarbonate, thiocyanate andascorbate at concentrations and in forms designed to replenish theirratios in the airway surface liquid to that associated with health. Itis also a focus of this invention that all of these systems need to worktogether to assure a healthy homeostasis and that the ratios of theseselected ions are critical to the orchestration of the host beneficialactions of these potentially host toxic factors. It follows thatreplenishing only one of the anions without consideration of the othersmay further disrupt the imbalance between these systems and exacerbatethe disease process.

The airway pathology of the cystic fibrosis patient provides evidencefor the critical roles of the ionic composition of exocrine secretions(airway surface liquid) for the functional maintenance of a mucosalsurface. All of these patients have mutations in both alleles of a genefor cystic fibrosis transmembrane conductance regulator (CFTR) thatfunctions as a precisely regulated anion channel to control ion andfluid homeostasis in the secretions that bathe epithelial surfaces. Inaddition to the initially identified chloride and associated sodiumimbalances, secretions from these subjects have variously been reportedto be deficient in glutathione, bicarbonate and thiocyanate, all ofwhich have been directly attributed to the lack of CFTR function. Thereare also several other deficiencies and dysfunctions that likely are theindirect consequence of the loss of anion transport. These include,among others, ascorbate deficiency, insufficiencies in pancreaticenzymes, dysfunctions in mucins, and reduction of inducible nitric oxidesynthase expression by airway epithelial cells and a consequentdiminution of exhaled nitric oxide. The lack of function of the singlegene product of cftr results in dehydrated accumulation ofmacromolecules, mucus plugging of ducts and blocking of delivery ofmacromolecules and interference with cilia function. It also results indysregulation of inflammation resulting in an inappropriate infiltrationof massive numbers of neutrophils and consequent overproduction ofreactive oxygen species (ROS) and reactive nitrogen intermediates (RNI).There is also a predisposition to infection of the airwaycharacteristically with opportunistic Pseudomonas aeruginosa thatultimately leads to morbidity and mortality in the majority of CFpatients. This species apparently utilizes the overproduction of RNI bythe host inflammatory response to drive nitrate respiration necessitatedby the reduction of oxygen in the inflammatory environment.

While the loss of CFTR function can be ascribed to the etiology of CF,there is also evidence that CFTR function may be compromised in otherchronic inflammatory lung diseases. It is the target of this inventionto restore the ionic composition in these airways to affectdown-regulation of the inflammatory response, suppression of neutrophilrecruitment, redirection of host destructive ROS and RNI to species withmore selective antimicrobial activity, altering the growth supportiveenvironment for CF pathogens, and to provide critical cofactors for thefunction of innate defense factors including lactoperoxidase andlactoferrin.

This will be accomplished by inhaled delivery of nebulized liquidcontaining glutathione, bicarbonate, thiocyanate and ascorbate atconcentrations designed to ultimately replenish the levels to that foundin healthy airway surface liquid.

The central premise of this proposal is that the healthy airwaymaintains homeostasis through the ionic composition of the exocrinesecretions of the submucosal glands distributed along the conductingairways of humans. It is assumed that inhaled particulates and potentialpathogens would initially encounter the airway surface liquid and notthe underlying airway epithelium in a normally healthy individual. Thecomponents of these secretions make critical contributions both to thedefense of the airway surfaces against infectious challenges and to theregulation of destructive inflammatory responses. Failure of thisprimary defense manifests as lung disease usually with systemicmanifestations suggesting that regional pathologic airway inflammationwill adversely affect other organs and systems outside the lung. One ofthe primary regulators of this ionic balance is the cystic fibrosistransmembrane regulator (CFTR) protein. When CFTR is absent, downregulated, or impaired (as demonstrated in cystic fibrosis (CF), chronicobstructive pulmonary disease (COPD), and asthma), the airway surfaceliquid has improper concentrations of selected ions, such asglutathione, bicarbonate, and thiocyanate, that are necessary to thefunction of critical soluble innate defense factors in the airway,regionally in the lung and systemically in the host. The result is achronic inflammatory state within the airway that affects other systemsimportant to maintaining airway homeostasis and lung function (i.e.,ascorbate and inducible nitric oxide synthase on airway epithelialcells) and the general health of the afflicted individual. Thehypothesis of this proposal is that restoring key components of theairway surface liquid will reestablish the function of the innatedefense system within the airway, regionally in the lung and, in manydisease states, systemically in the affected individual and betherapeutic in a number of chronic and acute pulmonary diseases.

In addition to the key mechanical defenses that facilitate airwayclearance of potentially harmful inhaled particulates and microbes, theairway surface liquid normally also provides a complex chemical defenseby bathing the airway surface with exocrine secretion of solublemolecules that provide direct antibacterial activity and help regulatethe host inflammatory response to these insults. The airway surface andthe glandular ductal epithelia likely play important roles in thepost-production modulation of the composition of these glandularproducts through the function of the apical membrane protein CFTR. Inthe ideal defense, microorganisms would be neutralized and cleared bythe intrinsic antimicrobial molecules that bathe the airway surfacewithout need of a second line of defense and without cellular contact.Lactoferrin (LF), lactoperoxidase (LPO), lysozyme, mucins, and secretoryIgA are among the principal antimicrobial proteins found in exocrinesecretions that bathe mucosal surfaces, including the conductingairways. Both LF and LPO are dependent on molecules and anions that aredelivered to the airway surface liquid either by glandular ductalepithelial cells or by the ciliated epithelium lining the airways.Specifically, hydrogen peroxide is synthesized by the NADPH oxidasefamily member dual oxidase 2 (DUOX 2) expressed by ductal epithelialcells, while nitric oxide (NO.) production is contributed by theciliated epithelial cells that express inducible nitric oxide synthase(iNOS). Furthermore, there is evidence that CFTR is not only a channelfor chloride, but is critical to the transport of several anions,including bicarbonate (HCO3⁻), reduced glutathione (GSH),nitrosoglutathione (GSNO), and thiocyanate (SCN⁻), that havedemonstrated critical roles in the function of LF, LPO, mucins, H₂O₂,NO., and superoxide anion (O₂ ⁻). Several reports cite that variousexocrine secretions of cystic fibrosis patients and other airwayconditions with direct or indirect modifications in CFTR (e.g., COPD,asthma, IPF, ciliary dyskinesia, etc.) are deficient in GSH, HCO₃ ⁻,SCN⁻, NO, and/or ascorbate.

Cystic fibrosis transmembrane conductance regulator (CFTR) is amultidomain integral apical membrane glycoprotein member of theATP-binding cassette (ABC) that is required to control ion and fluidhomeostasis on epithelial surfaces. Dysfunctions in the CFTR epithelialanion channel are fundamental to the etiology of cystic fibrosis (CF), adisease in which all individuals clinically diagnosed have mutations inboth CFTR alleles. The absence of this precisely regulated anion channelactivity disrupts ionic and water homeostasis on exocrine epithelialsurfaces resulting in dehydrated accumulations of macromolecules. Whilethis occurs on most exocrine surfaces, there are two sites where thedysfunction has profound pathologic effect. In the pancreas there is afailure of bicarbonate-rich fluid and enzyme secretion that impairsintestinal digestion and nutrient absorption of fats and fat-solublevitamins with serious consequence. The second site is in the conductingairways of the lungs, where the loss of CFTR function results in viscousmucus accumulation that impairs intrinsic clearance. Together, the thickmucus and subsequent impaired clearance allows for the colonization andaccumulation of microorganisms that provoke damaging inflammatoryresponses, which lead to an overall loss in airway function. There arethree apparently paradoxical aspects to the pathologic progression thatdistinguishes CF from other inflammatory airway diseases. Unlike theplethora of pathogens associated with other states of compromisedimmunity, such as chronic neutropenia, chronic granulomatous disease,leukocyte adhesion deficiencies, or advanced AIDS, infection with asingle pathogen (most notably Pseudomonas aeruginosa) is responsible forthe vast majority of morbidity and mortality in cystic fibrosis.Furthermore, the unique environment of the CF airway results in thegenetic adaptation of P. aeruginosa from the environmentally acquired,initially LPS smooth, motile, and antibiotic susceptible isolates to amucoidy phenotype characterized by the over-production of anextracellular polysaccharide (alginate) with concomitant loss of theO-side chain addition to lipopolysaccharide (LPS). Additionally, thereare multiple other genetic adaptations that confer resistance to innateand acquired immune defenses and antibiotic therapy and permit thesenormally aerobic bacteria to grow in an environment with reduced oxygen.The transition to a mucoidy phenotype and its associated genotypicmodifications generally heralds a severe decline in the health status ofthe CF patient.

The second feature unique to the CF airway is the chronic presence ofneutrophils on the airway surface, even in the apparent absence ofestablished infection. Normally, when neutrophils are recruited into theairway, NO produced by inducible nitric oxide synthase (iNOS) acts in anegative feedback loop to prevent neutrophil sequestration in theairway. However, in CF, the epithelial cells of the respiratory treehave been shown to be deficient in the expression of iNOS compared tonon-CF airways. iNOS expression has been shown to be mediated by reducedand oxidized glutathione (GSH and GSSG, respectively). Because CFTR isthe primary transporter of GSH, CF patients, who are characterized bycomplete deficiency or severely malfunctioning CFTR, do not have GSH orGSSG in their airway surface liquid; therefore, lacking CFTR leads todecreased levels of GSH and subsequent down-regulated iNOS expression.Thus, the airway has decreased NO concentrations leading to chronicneutrophil recruitment and sequestration within the CF lung. Studiesdemonstrate that when exogenous reducing equivalents are restored in theairway, epithelial iNOS expression increases.

In contrast to the deficiencies in iNOS in airway epithelial cells, CFpatients are not deficient in iNOS expression by professionalphagocytes, including monocytes and neutrophils. In comparison to non-CFsubjects with similar respiratory inflammation, CF patients havedemonstrably decreased exhaled NO. The airway surface liquid of CFpatients contains significant quantities of NO products, includingnitrate, nitrite, nitrosotyrosine, and peroxynitrite. These data wouldsuggest that there is NO being produced either by alternate forms of NOS(eNOS and/or nNOS) or more likely by iNOS from inflammatory cells in thearea. There is strong evidence that the neutrophils in the submucosaexpress significant quantities of iNOS. It is also probable that theneutrophils on the mucosal surface, as well as themonocytes/macrophages, alveolar macrophages, and mast cells, contributeto the production of NO products. In contrast to the epithelial cells,these inflammatory cells would characteristically also produce O₂ ⁻,H₂O₂ and myeloperoxidase, all of which would serve to further oxidizeNO, especially in the absence of antioxidants (ostensibly GSH) in themucosal milieu. While the peroxynitrite would theoretically be moretoxic than NO or GSNO, it would also be more readily converted tonitrate, nitrotyrosine and nitrosamines. It is clear that P. aeruginosathat lack nitric oxide reductase are incapable of growing by nitraterespiration due presumably to the toxicity of the nitric oxide generatedduring nitrate metabolism. It is also evident that there is adaptationof the bacteria that is necessary for optimum nitrate respiration andthat this adaptation results in increased resistance to ROS and RNI.However, P. aeruginosa is sensitive to H₂O₂ and NO adducts, includingnitrosoglutathione (GSNO) and NONOate, as well as nitrite. Furthermore,it is evident that the addition of GSH to GSNO or nitrite results in amore potent antibacterial specie, presumably from enhanced release ofNO. proximal to the bacterial target, suggesting that P. aeruginosa isvulnerable to NO. toxicity. At the same time, P. aeruginosa is dependenton NO products for growth in the absence of oxygen. The interactionsbetween O₂, NO., GSH, GSSG, H₂O₂, HCO₃ ⁻, and the enzymes or moleculesthat depend on these for function are complex.

Inducible NOS associated with phagocytic cells has been suggested toserve antimicrobial function through the generation of NO that reactswith O₂ ⁻, also generated by the phagocytic cell, to yield the reactivespecies peroxynitrite (ONOO⁻). Ideally this occurs within a phagosomedelivered to the target surface and not to the extracellular environmentwhere it might damage host tissues and lose effectiveness against itstarget. In contrast, the NO generated by the iNOS of ciliated epithelialcells apparently is released to the airway surface environment, but inthe presence of CFTR transported glutathione. There is evidence that NOreacts with glutathione to form the S-nitrosothiol nitrosoglutathione(GSNO), which is likely the biologically active species at the non-CFairway surface and not the NO that is delivered to the phagosome of theprofessional phagocyte. It is also clear that the GSNO is in thepresence of excess GSH in the normally healthy airway. Our preliminarydata indicate that GSH acts to limit the ability of P. aeruginosa toutilize nitrate, nitrite, or nitric oxide to grow under anaerobicconditions. Furthermore, nitrite and nitric oxide in the presence of GSHinhibit the aerobic growth of P. aeruginosa. Our data have alsodemonstrated that the anti-pseudomonal activities of GSNO and othernitric oxide donors are potentiated in the presence of exogenousglutathione. In addition, there are reports that both LF and LPO caninteract with NO to generate more potent antimicrobial species.

Clinical and experimental evidence suggest that the unique environmentpresented by the CF airways selectively permits the growth of P.aeruginosa. Evidence suggests that the airway environment of the CF lungwhere P. aeruginosa grows is anaerobic/microaerophilic. In order for P.aeruginosa to exhibit exponential growth as is evident on the CF airwaysurface, there must be an alternative to oxygen dependent metabolism. P.aeruginosa is capable of an eight electron reduction of nitrate tonitrogen. This nitrate respiration supports excellent growth of selectedstrains of P. aeruginosa under conditions of oxygen limitation. It isproposed that the inflammatory character that results in the airwaymucosa of the CF respiratory tree generates nitrate in a form andquantity sufficient to select for and support the growth of P.aeruginosa. Recent studies suggest that CFTR may play an important rolein the transport of the biological reductant reduced glutathione (GSH)to the airway surface liquid (ASL). Further evidence suggests that theASL of the CF patient is deficient in GSH compared to that of non-CFairways. This deficiency would certainly result in a reduction in theantioxidant capacity operating on airway surfaces and predispose thesesurfaces to oxidative damage. It is further the contention of thisproposal that the deficiency in secreted glutathione results in adiminished capacity of the ASL to inhibit P. aeruginosa fromestablishing an infection at these mucosal surfaces. It is proposed thatglutathione (either GSH or GSSG) is critical to the antimicrobialfunction of NO and that the normally healthy airway generates an NOadduct of glutathione (S-nitrosoglutathione, GSNO) that serves both todeliver NO in an antibacterial form and to limit the conversion of NO tonitrate, thus restricting the availability of this growth-criticalsubstrate. The antimicrobial properties of the airway surface liquid arethus facilitated by the production of NO by the airway epithelial cellsthat in non-CF subjects express iNOS.

LF has the ability of high affinity binding of two ferric ions incoordination with the binding of carbonate. Four of the coordinate sitesof iron are associated with amino acid residues in each of the pair ofiron binding clefts in the N- and C-terminal lobes of the LF protein.The remaining two coordinate sites of ferric ion are occupied by two ofthe coordinate sites of carbonate while the third coordinate site ofcarbonate is bound to its amino acid residue in the LF lobes. Thissynergistic binding results in an unusually high affinity resulting instabilization of the iron in the ferric transition state. Other anions(e.g., HCO₃ ⁻) are also capable of fitting in to the anion coordinatesite of LF, but can only occupy one of the two free coordinate sites ofiron. The remaining free coordinate site can then participate in aHaber-Weiss interaction in the presence of H₂O₂, generating hydroxylradical. This reaction could be amplified if there is reducingpotential, such as O₂ ⁻ or ascorbate, available to cycle the iron to aferrous state. In addition to the reducing potential of ascorbate, it isof a size and configuration that it fits in the anion coordinate sitesof lactoferrin, limiting stabilized carbonate-Fe³⁺ binding. If generatedproximal to a susceptible bacterial surface, it would kill potentialpathogens, conversely it could also damage host tissues. Therefore itwould be predicted that iron binding by LF in coordination withcarbonate would scavenge iron and serve an antioxidant function. Incontrast, bicarbonate and ascorbate would serve to facilitate thegeneration of reactive oxygen species and bactericidal activity on atarget pathogen surface. Therefore the ratio of carbonate tobicarbonate, especially in the presence of other species such asascorbate, O₂ ⁻, H₂O₂ and NO, may serve an important regulatory functionin determining the antibacterial vs. antioxidant effects of LF.

Furthermore, bicarbonate is transported into the mucosa via CFTR;therefore, CF patients lack bicarbonate within their ASL, which impairsLF's activity. Additionally, our preliminary studies have shown thatbicarbonate inhibits the alginate production associated with thedetrimental emergence of the mucoidy phenotype of P. aeruginosa. Furtherhindering LF activity is the chronic inflammatory state within the CFairway that depletes already low reserves of ascorbate. Ascorbate alsoworks with GSH to maintain a reduced environment, as well as maintainproper mucocilliary clearance. Thus, restoring bicarbonate and ascorbateto the airway surface can aid in LF activity and stymie the developmentand/or expression of mucoidy P. aeruginosa.

LPO catalyzes the oxidation of SCN⁻ by H₂O₂ to form hypothiocyanite(OSCN⁻), a potent antimicrobial species that works by oxidizingessential sulfhydryls of target proteins on bacterial surfaces. This caneffectively inhibit bacterial metabolism through neutralization ofenzymes such as hexose kinases necessary for the transport of sugars.Unlike its neutrophil counterpart myeloperoxidase (MPO), LPO cannot usethe halide Cl⁻ as substrate and does not generate the potentially hostnoxious product hypochlorous acid (HOCl). While having potentantimicrobial activity, OSCN⁻ has the added advantage that it isrelatively innocuous to host tissues. Therefore in the presence ofadequate concentrations of SCN⁻ and functioning LPO, there would becompetition for H₂O₂ that would limit the activity of MPO generation ofHOCl. There is also evidence that MPO may use SCN⁻ in preference to Cl.Conversely, limitations in SCN⁻ would serve to favor the lessdiscriminating and more reactive HOCl.

Therefore, restoring CFTR transported glutathione, HCO₃, and SCN⁻ andenhancing the reducing potential in the CF airway by adding ascorbateinto the airway, will reestablish the ASL to a more “normal” ionicbalance that is suited to restore proper function of natural defenses,such as LF and LPO activity, and thus, will slow the progression of CFand several other lung diseases. Replacing these elements with uniqueinhaled formulations will enhance homeostatic mechanisms required formucociliary clearance and other host-sparing antibacterial functions andwill reduce the pathologic inflammatory elements that uncontrolleddamage the host by accelerating lung function deterioration favoring P.aeruginosa growth.

The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein. Allpublications, patent applications, patents, patent publications, andother references cited herein are incorporated by reference in theirentireties for the teachings relevant to the sentence and/or paragraphin which the reference is presented.

That which is claimed is:
 1. A method for treating a pulmonary or airwaydisorder or disease by inhibiting microbial growth in an airway of asubject in need thereof, comprising administering to the airway of thesubject an effective amount of a composition comprising: a) aglutathione, or a pharmaceutically-acceptable salt of glutathione; b) anorganic acid or a pharmaceutically-acceptable salt of an organic acid;and c) a bicarbonate salt, wherein the pulmonary or airway disorder ordisease is selected from the group consisting of chronic inflammatorylung disease, inflammation and/or infection associated with lungtransplantation, acute lung rejection, asthma, cystic fibrosis, chronicobstructive pulmonary disease (COPD) and any combination thereof,thereby treating the pulmonary or airway disorder or disease byinhibiting microbial growth in the airway of the subject.
 2. The methodof claim 1, wherein the organic acid is ascorbic acid.
 3. The method ofclaim 1, wherein the bicarbonate salt is sodium bicarbonate or potassiumbicarbonate.
 4. The method of claim 1, wherein the amount of each ofcomponent (a), (b) and (c) of the composition is present such that theamount of bicarbonate salt results in a pH in a range from about 5 toabout 9, if in an aqueous solution, and wherein the bicarbonate salt ispresent in a molar amount equal to the combined molar amount of theglutathione and the organic acid or in a molar excess of the combinedmolar amount of the glutathione and the organic acid.
 5. The method ofclaim 4, wherein the molar excess is greater than 1.0 and less thanabout 1.5.
 6. The method of claim 1, wherein the composition furthercomprises from about 0.01% to about 5% by weight of apharmaceutically-acceptable thiocyanate salt.
 7. The method of claim 1,wherein the composition is in the form of a particle.
 8. The method ofclaim 7, wherein the particle is mixed with a gas or liquid propellantfor use in inhalation therapy.
 9. The method of claim 1, comprising thefurther step of administering to the subject an effective amount of atherapeutic agent.
 10. The method of claim 9, wherein the therapeuticagent is an inhaled corticosteroid (ICS) or bronchodilator.
 11. Themethod of claim 9, wherein the therapeutic agent is selected from thegroup consisting of Fluticasone, Budesonide, Mometasone, Ciclesonide,Flunisolide, Beclomethasone, Albuterol, Levalbuterol, Ipratropium,Tiotropium, Formoterol, Arformoterol, Indacaterol, Aclidinium,Pirbuterol and any combination thereof.
 12. The method of claim 1,wherein the glutathione is oxidized glutathione.
 13. The method of claim1, wherein the glutathione is reduced glutathione.
 14. The method ofclaim 1, wherein upon administration of the composition to the subject,the concentration of glutathione in the airway surface liquid of thesubject is from about 0.1 mM to about 1.0 mM.
 15. The method of claim 6,wherein upon administration of the composition to the subject, theconcentration of thiocyanate in the airway surface liquid of the subjectis from about 0.5 mM to about 3.0 mM.
 16. A method of restoringhomeostasis to and/or maintaining homeostasis in a mucosal membrane of asubject with a pulmonary or airway disorder or disease by inhibitingmicrobial growth in an airway of the subject, comprising administeringto the airway of the subject an effective amount of a compositioncomprising: a) a glutathione, or a pharmaceutically-acceptable salt ofglutathione; b) an organic acid, or a pharmaceutically-acceptable saltof an organic acid; and c) a bicarbonate salt, wherein the pulmonary orairway disorder or disease is selected from the group consisting ofchronic inflammatory lung disease, inflammation and/or infectionassociated with lung transplantation, acute lung rejection, asthma,cystic fibrosis, chronic obstructive pulmonary disease (COPD) and anycombination thereof, thereby restoring homeostasis to and/or maintaininghomeostasis in the mucosal membrane of the subject by inhibitingmicrobial growth in the airway of the subject.
 17. The method of claim16, wherein the organic acid is ascorbic acid.
 18. The method of claim16, wherein the bicarbonate salt is sodium bicarbonate or potassiumbicarbonate.
 19. The method of claim 16, wherein the composition furthercomprises from about 0.01% to about 5% by weight of apharmaceutically-acceptable thiocyanate salt.
 20. A method of inhibitingmicrobial growth in an airway of a subject, comprising administering tothe airway of the subject an effective amount of a compositioncomprising: a) a glutathione, or a pharmaceutically-acceptable salt ofglutathione; b) an organic acid, or a pharmaceutically-acceptable saltof an organic acid; and c) a bicarbonate salt, thereby inhibitingmicrobial growth in the airway of the subject.
 21. The method of claim20, wherein the organic acid is ascorbic acid.
 22. The method of claim20, wherein the bicarbonate salt is sodium bicarbonate or potassiumbicarbonate.
 23. The method of claim 20, wherein the composition furthercomprises from about 0.01% to about 5% by weight of apharmaceutically-acceptable thiocyanate salt.
 24. The method of claim 1,wherein the composition is in the form of a solid formulation.
 25. Themethod of claim 16, wherein the composition is in the form of a solidformulation.
 26. The method of claim 20, wherein the composition is inthe form of a solid formulation.